Production of 3-hydroxypropionic acid using beta-alanine/pyruvate aminotransferase

ABSTRACT

Methods of using beta-alanine/pyruvate aminotransferase to produce 3-hydroxypropionic acid and derivatives thereof, from beta-alanine, are disclosed. Cells and recombinant nucleic acids that can be used to practice the methods are also disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This is the U.S. National Stage of International Application No.PCT/US2004/040827, filed Dec. 6, 2004 (published in English under PCTArticle 21(2)), which claims the benefit of U.S. Provisional ApplicationNo. 60/527,357, filed Dec. 4, 2003, herein incorporated by reference inits entirety.

FIELD

This disclosure relates to cells having beta-alanine/pyruvateaminotransferase activity that can be used to convert beta-alanine to3-hydroxypropionic acid (3-HP) and other organic compounds.

BACKGROUND

Organic chemicals such as organic acids, esters, and polyols can be usedto synthesize plastic materials and other products. To meet theincreasing demand for organic chemicals, more efficient andcost-effective production methods are being developed which utilize rawmaterials based on carbohydrates rather than hydrocarbons. For example,certain bacteria have been used to produce large quantities of lacticacid used in the production of polylactic acid.

3-hydroxypropionic acid (3-HP) is an organic acid. Several chemicalsynthesis routes have been described to produce 3-HP, and biocatalyticroutes have also been disclosed (WO 01/16346 to Suthers et al.). 3-HPhas utility for specialty synthesis and can be converted to commerciallyimportant intermediates by known art in the chemical industry, such asacrylic acid by dehydration, malonic acid by oxidation, esters byesterification reactions with alcohols, and 1,3-propanediol byreduction.

SUMMARY

The compound 3-hydroxypropionic acid (3-HP) can be produced bybiocatalysis from beta-alanine (FIG. 1). Beta-alanine can be synthesizedin cells from carnosine, beta-alanyl arginine, beta-alanyl lysine,uracil via 5,6-dihydrouracil and N-carbamoyl-beta-alanine,N-acetyl-beta-alanine, anserine, or aspartate. However, these routes arerelatively inefficient because they require rare precursors or startingcompounds that are more valuable than 3-HP. As an alternative,beta-alanine can be produced from alpha-alanine by an enzyme havingalanine 2,3-aminomutase activity (FIG. 1), such as SEQ ID NOS: 22, 24,and 26, as well as variants, fragments, and fusions thereof that retainalanine 2,3-aminomutase activity. A novel alanine 2,3-aminomutasenucleic acid sequence (SEQ ID NO: 21) and corresponding amino acidsequence (SEQ ID NO: 22), as well as variants, fragments, fusions, andpolymorphisms thereof that retain alanine 2,3-aminomutase activity, isdisclosed.

Also disclosed are methods of producing 3-HP from beta-alanine usingbeta-alanine/pyruvate aminotransferase (BAPAT) sequences. In oneexample, a BAPAT peptide is a sequence that includes SEQ ID NO: 18 or20, or variants, fragments, or fusions thereof that retain BAPATactivity. Exemplary BAPAT nucleic acid sequences include, but are notlimited to, SEQ ID NO: 17 or 19. BAPAT sequences can be used totransform cells, such that the transformed cells have BAPAT activity,which allows the cells to produce 3-HP from beta-alanine through amalonate semialdehyde (3-oxopropanoate) intermediate.

Transformed cells having BAPAT activity, which allows the cell toconvert beta-alanine to 3-HP through a malonate semialdehydeintermediate, are disclosed. Such cells can be eukaryotic or prokaryoticcells, such as yeast cells, plant cells, fungal cells, or bacterialcells such as Lactobacillus, Lactococcus, Bacillus, or Escherichiacells. A particular example of such cells were deposited with theAmerican Type Culture Collection (Manassas, Va.) on Dec. 6, 2004(Accession No. PTA-6411). In one example, the cell is transformed with aBAPAT nucleic acid sequence that confers to the transformed cells BAPATactivity.

One aspect of the disclosure provides transformed cells, which inaddition to BAPAT activity (EC 2.1.6.18), include other enzymeactivities, such as 3-hydroxypropionate dehydrogenase activity (EC1.1.1.59), lipase or esterase activity (EC 3.1.1.-), aldehydedehydrogenase activity (EC 1.2.1.-), alcohol dehydrogenase activity (EC1.1.1.1), or combinations thereof. In particular examples, the cell canfurther include alanine 2,3-aminomutase activity. Accordingly, thedisclosure also provides methods of producing one or more of theseproducts. These methods involve culturing the cell that includes3-hydroxypropionate dehydrogenase activity, lipase or esterase activity,alanine 2,3-aminomutase activity, aldehyde dehydrogenase activity,alcohol dehydrogenase activity, or combinations thereof, underconditions that allow the product to be produced.

The disclosed cells can be used to produce nucleic acid molecules,peptides, and organic compounds. In one example the disclosed cells areused in a culture system to produce large quantities of 3-HP andderivatives thereof such as 1,3-propanediol, polymerized 3-HP,co-polymers of 3-HP, and esters of 3-HP. 3-HP is both biologically andcommercially important. For example, the nutritional industry can use3-HP as a food, feed additive or preservative, while the derivativesmentioned above can be produced from 3-HP.

A production cell having at least one exogenous nucleic acid encoding abeta-alanine/pyruvate aminotransferase, is disclosed. Such productioncells can be used to produce 3-HP or derivatives thereof. In oneexample, the nucleic acid sequence includes SEQ ID NO: 17 or 19 (orfragments, variants, or fusions thereof that retain BAPAT activity).Production cells can be used to express peptides that have an enzymaticactivity, such as beta-alanine/pyruvate aminotransferase,3-hydroxypropionate dehydrogenase activity, lipase or esterase activity,alanine 2,3-aminomutase activity, aldehyde dehydrogenase activity,alcohol dehydrogenase activity, or combinations thereof. Methods ofproducing peptides encoded by the nucleic acid sequences described aboveare disclosed.

Several methods of producing 3-HP from beta-alanine using the disclosedcells having BAPAT activity are disclosed. In one example, the cell istransfected with one or more enzymes necessary to convert 3-HP frombeta-alanine. In another example, the method includes purifyingbeta-alanine from the cell, then contacting the beta-alanine withpeptides necessary to convert 3-HP from beta-alanine.

In some examples, products are produced in vitro (outside of a cell). Inother examples, products are produced using a combination of in vitroand in vivo (within a cell) methods. In yet other examples, products areproduced in vivo. For methods involving in vivo steps, the cells can beisolated cultured cells or whole organisms such as transgenic plants orsingle-celled organisms such as yeast and bacteria. Such cells arereferred to as production cells. Products produced by these productioncells can be organic products such as 3-HP and derivatives thereof suchas an ester of 3-HP, polymerized 3-HP, or 1,3-propanediol.

Recombinant nucleic acids that can be used to generate the productioncells and practice the methods disclosed herein are provided. Forexample, operons including two or more nucleic acid sequences, such astwo, three, four, five, six, or even seven sequences, each encoding apeptide needed for the production of 3-HP from beta-alanine aredisclosed. In a particular example, the recombinant nucleic acidsequence includes a sequence encoding a beta-alanine/pyruvateaminotransferase and a nucleic acid sequence encoding a dehydrogenasecapable of converting malonate semialdehyde to 3-HP, such as3-hydroxypropionate dehydrogenase. Such recombinant nucleic acidsequences can further include a nucleic acid sequence that encodes analanine 2,3-aminomutase, lipase or esterase, aldehyde dehydrogenase,alcohol dehydrogenase, or combinations thereof. In addition, recombinantnucleic acid sequences can additionally include one or more promotersequences to drive expression of the coding sequence. The disclosednucleic acids can be incorporated into a vector, which can be used totransform a cell, or be incorporated into the genome of the cell, orboth.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a diagram of a pathway for generating 3-HP and derivativesthereof via beta-alanine.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

SEQ ID NOS: 1 and 2 are PCR primers used to PCR amplify a CAT gene.

SEQ ID NOS: 3 and 4 are PCR primers used to confirm insertion of a CATgene into the ldhA locus.

SEQ ID NOS: 5 and 6 are PCR primers used to PCR amplify abeta-alanine/pyruvate aminotransferase gene from P. aeruginosa.

SEQ ID NOS: 7 and 8 are PCR primers used to PCR amplify a P. putidabeta-alanine/pyruvate aminotransferase gene.

SEQ ID NOS: 9 and 10 are PCR primers used to PCR amplify an mmsB genefrom P. aeruginosa.

SEQ ID NOS: 11 and 12 are PCR primers used to PCR amplify an mmsB genefrom pET28-mmsB.

SEQ ID NOS: 13 and 14 are PCR primers used to PCR amplify an alanine2,3-aminomutase gene.

SEQ ID NOS: 15 and 16 are PCR primers used to PCR amplify a P. putidabeta-alanine/pyruvate aminotransferase gene from pPRO-PpBAPAT.

SEQ ID NO: 17 is a nucleic acid sequence of a beta-alanine/pyruvateaminotransferase DNA from P. putida.

SEQ ID NO: 18 is an amino acid sequence encoded by SEQ ID NO: 17.

SEQ ID NO: 19 is a nucleic acid sequence of a beta-alanine/pyruvateaminotransferase DNA from Pseudomonas aeruginosa.

SEQ ID NO: 20 is an amino acid sequence encoded by SEQ ID NO: 19.

SEQ ID NOS: 21, 23, and 25 are alanine 2,3-aminomutase nucleic acidsequences. The corresponding amino acid sequences are shown in SEQ IDNOS: 22, 24 and 26, respectively.

SEQ ID NO: 27 is a nucleic acid sequence of a3-hydroxypropionate/3-hydroxyisobutyrate dehydrogenase (mmsB) DNA.

SEQ ID NO: 28 is an amino acid sequence encoded by SEQ ID NO: 27.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Abbreviations and Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. As used herein,“comprising” means “including” and the singular forms “a” or “an” or“the” include plural references unless the context clearly dictatesotherwise. For example, reference to “comprising a protein” includes oneor a plurality of such proteins, and reference to “comprising the cell”includes reference to one or more cells and equivalents thereof known tothose skilled in the art, and so forth. The term “or” refers to a singleelement of stated alternative elements or a combination of two or moreelements, unless the context clearly indicates otherwise. For example,the phrase “lipase activity or esterase activity” refers to lipaseactivity, esterase activity, or a combination of both lipase activityand esterase activity.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features andadvantages of the disclosure are apparent from the following detaileddescription and the claims.

Alanine 2,3-aminomutase: An enzyme which can convert alpha-alanine tobeta-alanine, for example in a cell. Includes any alanine2,3-aminomutase gene, cDNA, RNA, or protein from any organism, such as aprokaryote. In particular examples, an alanine 2,3-aminomutase nucleicacid sequence includes the sequence shown in SEQ ID NOS: 21, 23 or 25,as well as fragments, variants, or fusions thereof that retain theability to encode a protein having alanine 2,3-aminomutase activity. Inanother example, an alanine 2,3-aminomutase protein includes the aminoacid sequence shown in SEQ ID NO: 22, 24 or 26, as well as fragments,fusions, or variants thereof that retain alanine 2,3-aminomutaseactivity.

An alanine 2,3-aminomutase amino acid sequence includes a full-lengthsequence, such as SEQ ID NO: 22, 24 or 26, as well as shorter sequenceswhich retain the ability to convert alpha-alanine to beta-alanine, suchas amino acids 50-390 of SEQ ID NO: 22 or 24, amino acids 101-339 of SEQID NO: 22 or 24, amino acids 15-390 of SEQ ID NO: 26, and amino acids15-340 of SEQ ID NO 26. This description includes alanine2,3-aminomutase allelic variants, as well as any variant, fragment, orfusion sequence which retains the ability to convert alpha-alanine tobeta-alanine.

Examples of alanine 2,3-aminomutase fragments which can be used include,but are not limited to: amino acids 50-390, 50-350, 60-350, 75-340, or100-339 of SEQ ID NO: 22 or 24 and amino acids 1-390, 15-390, 15-340 or19-331 of SEQ ID NO: 26.

Alanine 2,3-aminomutase activity: The ability of an alanine2,3-aminomutase to convert alpha-alanine to beta-alanine. In oneexample, such activity occurs in a cell. In another example, suchactivity occurs in vitro. Such activity can be measured using any assayknown in the art. For example, alanine 2,3-aminomutase activity can beidentified by incubating the enzyme with either alpha-alanine orbeta-alanine and determining the reaction products by high-performanceliquid chromatography (for example using the method of Abe et al. J.Chromatography B, 712:43-9, 1998).

Examples of substitutions which can be made, while still retainingalanine 2,3-aminomutase activity, include, but are not limited to: V21Ior V21L; Y71P; L17I; K361R; A410V; and/or Y430F or Y430W of SEQ ID NO:22 or 24, and T40S; V96I or V96L; D102E; A252V; or L393V of SEQ ID NO:26, as well as combinations thereof.

Beta-alanine/pyruvate aminotransferase (BAPAT): An enzyme that canconvert beta-alanine and pyruvate to malonate semialdehyde plus alanine.Includes any beta-alanine/pyruvate aminotransferase gene, cDNA, RNA, orprotein from any organism, such as a prokaryote or eukaryote. Thisdescription includes beta-alanine/pyruvate aminotransferase allelicvariants, as well as any variant, fragment, or fusion protein sequencewhich retains the ability to convert beta-alanine and pyruvate tomalonate semialdehyde plus alanine.

Examples include, but are not limited to, any beta-alanine/pyruvateaminotransferase gene, cDNA, RNA, or protein from Pseudomonas aeruginosaPAO1 (for example GenBank Accession Nos: AE004451.1 (nucleic acid) andAAG03522 (protein)); Pseudomonas putida IFO 14796 (for example GenBankAccession No. P28269 protein); P. putida KT2440 (for example GenBankAccession No. NC_(—)002947); Arabidopsis thaliana (for example GenBankAccession Nos: AY085348.1 (nucleic acid) and AAM62579 (protein)); rat(for example GenBank Accession Nos: NM_(—)031835 (nucleic acid) andNP_(—)114023 (protein)); and Xenopus laevis (for example GenBankAccession No. BE507883).

In particular examples, a beta-alanine/pyruvate aminotransferase nucleicacid sequence includes the sequence shown in SEQ ID NOS: 17 or 19, aswell as fragments, variants, or fusions thereof that retain the abilityto encode a peptide having beta-alanine/pyruvate aminotransferaseactivity. In another example, a beta-alanine/pyruvate aminotransferaseprotein includes the amino acid sequence shown in SEQ ID NO: 18 or 20,as well as fragments, fusions, or variants thereof that retainbeta-alanine/pyruvate aminotransferase activity.

Beta-alanine/pyruvate aminotransferase activity. The ability of abeta-alanine/pyruvate aminotransferase to convert beta-alanine andpyruvate to malonate semialdehyde plus alanine. Includes members of EC2.6.1.18. In one example, such activity occurs in a cell. In anotherexample, such activity occurs in vitro. Such activity can be measuredusing any assay known in the art. For example, beta-alanine/pyruvateaminotransferase activity can be identified by incubating the enzymewith beta-alanine and pyruvate and determining the reaction products bypaper chromatography, amino acid analysis (Yonaha et al., FEBS Letts.71: 21-24, 1976), reaction with an aldehyde-specific reagent (Waters andVenables, FEMS Microbiol. Letts. 34: 279-282, 1986), or by coupling theproduction of malonate semialdehyde to its reduction by aNAD(P)H-dependent 3-HP dehydrogenase and monitoring the change in A₃₄₀.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences which determinetranscription. cDNA can be synthesized by reverse transcription frommessenger RNA extracted from cells.

Conservative substitution: One or more amino acid substitutions (forexample 1, 2, 5 or 10 amino acid residues) for amino acid residueshaving similar biochemical properties. Typically, conservativesubstitutions have little to no impact on the activity of a resultingpeptide. For example, a conservative substitution is an amino acidsubstitution in a beta-alanine/pyruvate aminotransferase peptide thatdoes not substantially affect the ability of the peptide to convertbeta-alanine to malonate semialdehyde. In a particular example, aconservative substitution is an amino acid substitution in abeta-alanine/pyruvate aminotransferase peptide, such as a conservativesubstitution in SEQ ID NO: 18 or 20, that does not significantly alterthe ability of the protein to convert beta-alanine to malonatesemialdehyde, or other downstream products such as 3-HP.

An alanine scan can be used to identify amino acid residues in abeta-alanine/pyruvate aminotransferase peptide that can toleratesubstitution. In one example, beta-alanine/pyruvate aminotransferaseactivity is not altered by more than 25%, for example not more than 20%,for example not more than 10%, when an alanine, or other conservativeamino acid (such as those listed below), is substituted for one or morenative amino acids.

In a particular example, beta-alanine/pyruvate aminotransferase activityis not substantially altered if the amount of 3-HP produced is notreduced by more than about 25%, such as not more than about 10%, than anamount of 3-HP production in the presence of a beta-alanine/pyruvateaminotransferase containing one or more conservative amino acidsubstitutions, as compared to an amount of 3-HP production in thepresence of a native beta-alanine/pyruvate aminotransferase.

A peptide can be produced to contain one or more conservativesubstitutions by manipulating the nucleotide sequence that encodes thatpeptide using, for example, standard procedures such as site-directedmutagenesis or PCR. Alternatively, a peptide can be produced to containone or more conservative substitutions by using standard peptidesynthesis methods.

Substitutional variants are those in which at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. Examples of amino acids which may be substituted for anoriginal amino acid in a protein and which are regarded as conservativesubstitutions include: Ser for Ala; Lys for Arg; Gln or His for Asn; Glufor Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro for Gly, Asn or Glnfor His; Leu or Val for Ile; Ile or Val for Leu; Arg or Gln for Lys; Leuor Ile for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyrfor Trp; Trp or Phe for Tyr; and Ile or Leu for Val.

Further information about conservative substitutions can be found in,among other locations in, Ben-Bassat et al., (J. Bacteriol. 169:751-7,1987), O'Regan et al., (Gene 77:237-51, 1989), Sahin-Toth et al.,(Protein Sci. 3:240-7, 1994), Hochuli et al., (Bio/Technology 6:1321-5,1988), WO 00/67796 (Curd et al.) and in standard textbooks of geneticsand molecular biology.

Dehydrogenase: An enzyme that oxidizes a substrate by transferringhydrogen to an acceptor, such as NAD/NADP and also catalyzes the reversereaction.

Deletion: The removal of a sequence of a nucleic acid, for example DNA,the regions on either side being joined together.

Detectable: Capable of having an existence or presence ascertained. Forexample, production of 3-HP from beta-alanine is detectable if thesignal generated from 3-HP is strong enough to be measured.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer which includesthe genetic material of most living organisms (some viruses have genesincluding ribonucleic acid, RNA). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides,referred to as codons, in DNA molecules code for amino acid in apeptide. The term codon is also used for the corresponding (andcomplementary) sequences of three nucleotides in the mRNA into which theDNA sequence is transcribed.

Exogenous: The term “exogenous” as used herein with reference to nucleicacid and a particular cell refers to any nucleic acid that does notoriginate from that particular cell as found in nature. Thus, anon-naturally-occurring nucleic acid is considered to be exogenous to acell once introduced into the cell. A nucleic acid that isnaturally-occurring also can be exogenous to a particular cell. Forexample, an entire chromosome isolated from cell X is an exogenousnucleic acid with respect to cell Y once that chromosome is introducedinto cell Y, even if X and Y are the same cell type.

Functionally Equivalent: Having a similar function. In the context of abeta-alanine/pyruvate aminotransferase molecule, functionally equivalentmolecules include different molecules that retain the function ofbeta-alanine/pyruvate aminotransferase. For example, functionalequivalents can be provided by sequence alterations in abeta-alanine/pyruvate aminotransferase, wherein the peptide with one ormore sequence alterations retains a function of the unaltered peptide,such that it retains its ability to convert beta-alanine to malonatesemialdehyde.

Examples of sequence alterations include, but are not limited to,conservative substitutions, deletions, mutations, frameshifts, andinsertions. In one example, a given peptide binds an antibody, and afunctional equivalent is a peptide that binds the same antibody. Thus afunctional equivalent includes peptides that have the same bindingspecificity as a peptide, and that can be used as a reagent in place ofthe peptide (such as in the production of 3-HP and derivatives thereof).In one example a functional equivalent includes a peptide wherein thebinding sequence is discontinuous, wherein the antibody binds a linearepitope. Thus, if the peptide sequence is NMPEHAGASL (amino acids 1-10of SEQ ID NO: 18) a functional equivalent includes discontinuousepitopes, that can appear as follows (**=any number of intervening aminoacids): NH₂-**-N**M**P**E**H**A**G**A**S**L-COOH. In this example, thepeptide is functionally equivalent to amino acids 1-10 of SEQ ID NO: 18if the three dimensional structure of the peptide is such that it canbind a monoclonal antibody that binds amino acids 1-10 of SEQ ID NO: 18.

Hybridization: To form base pairs between complementary regions of twostrands of DNA, RNA, or between DNA and RNA, thereby forming a duplexmolecule. Nucleic acid hybridization techniques can be used to identifyan isolated nucleic acid molecule within the scope of the disclosure.Briefly, a nucleic acid molecule having some homology to nucleic acidmolecules encoding one of the disclosed enzymes (such as homology to SEQID NOS: 17, 19, 21, or variants, fusions, or fragments thereof) can beused as a probe to identify a similar nucleic acid molecule byhybridization under conditions of moderate to high stringency. Onceidentified, the nucleic acid molecule then can be purified, sequenced,and analyzed to determine if it has the desired enzyme activity, such asBAPAT activity.

Hybridization can be done by Southern or Northern analysis to identify aDNA or RNA sequence, respectively, that hybridizes to a probe. The probecan be labeled, for example with a biotin, a fluorophore, digoxygenin,an enzyme, or a radioisotope such as ³²P. The DNA or RNA to be analyzedcan be electrophoretically separated on an agarose or polyacrylamidegel, transferred to nitrocellulose, nylon, or other suitable membrane,and hybridized with the probe using standard techniques well known inthe art such as those described in sections 7.39-7.52 of Sambrook etal., (1989) Molecular Cloning, second edition, Cold Spring HarborLaboratory, Plainview, N.Y. Typically, a probe is at least about 20nucleotides in length. For example, a probe including 20 contiguousnucleotides of a beta-alanine/pyruvate aminotransferase (such as 20contiguous nucleotides of SEQ ID NO:17 or 19) can be used to identify anidentical or similar nucleic acid. Probes longer or shorter than 20nucleotides can also be used.

The disclosure also provides isolated nucleic acid sequences that are atleast about 12 bases in length (such as at least about 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 40, 50, 60, 100, 250, 500, 750, 1000, 1400,2000, 3000, 4000, or 5000 bases in length) and hybridize, underhybridization conditions, to the sense or antisense strand of an enzymedisclosed herein. The hybridization conditions can be moderately orhighly stringent hybridization conditions.

Moderately stringent hybridization conditions are when the hybridizationis performed at about 42° C. in a hybridization solution containing 25mM KPO₄ (pH 7.4), 5×SSC, 5×Denhart's solution, 50 μg/mL denatured,sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 1-15ng/mL probe (about 5×10⁷ cpm/μg), while the washes are performed atabout 50° C. with a wash solution containing 2×SSC and 0.1% sodiumdodecyl sulfate (SDS).

Highly stringent hybridization conditions are when the hybridization isperformed at about 42° C. in a hybridization solution containing 25 mMKPO₄ (pH 7.4), 5×SSC, 5×Denhart's solution, 50 μg/mL denatured,sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 1-15ng/mL probe (about 5×10⁷ cpm/μg), while the washes are performed atabout 65° C. with a wash solution containing 0.2×SSC and 0.1% SDS.

3-hydroxypropionate dehydrogenase: An enzyme that can convert malonatesemialdehyde to 3-HP. Includes any 3-hydroxypropionate dehydrogenasegene, cDNA, RNA, or protein from any organism, such as a prokaryote oreukaryote. This description includes 3-hydroxypropionate dehydrogenaseallelic variants, as well as any variant, fragment, or fusion proteinsequence that retains the ability to convert malonate semialdehyde to3-HP.

Examples include, but are not limited to any gene, cDNA, RNA, or proteinfrom Pseudomonas aeruginosa encoding 3-hydroxypropionate dehydrogenase(mmsB gene, see Example 5). In particular examples, a mmsB nucleic acidsequence includes the sequence shown in SEQ ID NO: 27, as well asfragments, variants, or fusions thereof that retain the ability toencode a peptide having 3-hydroxypropionate dehydrogenase activity. Inanother example, a mmsB protein includes the amino acid sequence shownin SEQ ID NO: 28, as well as fragments, fusions, or variants thereofthat retain 3-hydroxypropionate dehydrogenase activity.

3-hydroxypropionate dehydrogenase activity: The ability of a3-hydroxypropionate dehydrogenase to convert malonate semialdehyde to3-HP. In one example, such activity occurs in a cell. In anotherexample, such activity occurs in vitro. Such activity can be measuredusing any assay known in the art. For example, 3-hydroxypropionatedehydrogenase activity can be identified by incubating the enzyme withmalonate semialdehyde plus NADH or NADPH and measuring the consumptionof NADH or NADPH by monitoring the decrease in absorbance at 340 nm.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule or protein) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, such as other chromosomal andextrachromosomal DNA and RNA, and proteins. Nucleic acid molecules andproteins that have been “isolated” include nucleic acid molecules andproteins purified by standard purification methods. The term alsoembraces nucleic acid molecules and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleic acidmolecules, proteins and peptides.

In one example, isolated refers to a naturally-occurring nucleic acidmolecule that is not immediately contiguous with both of the sequenceswith which it is immediately contiguous (one on the 5′ end and one onthe 3′ end) in the naturally-occurring genome of the organism from whichit is derived. For example, an isolated nucleic acid molecule can be,without limitation, a recombinant DNA molecule of any length, providedone of the nucleic acid sequences normally found immediately flankingthat recombinant DNA molecule in a naturally-occurring genome is removedor absent. Thus, an isolated nucleic acid includes, without limitation,a recombinant DNA that exists as a separate molecule (for example, acDNA or a genomic DNA fragment produced by PCR or restrictionendonuclease treatment) independent of other sequences as well asrecombinant DNA that is incorporated into a vector, an autonomouslyreplicating plasmid, a virus (for example, a retrovirus, adenovirus, orherpes virus), or into the genomic DNA of a prokaryote or eukaryote. Inaddition, an isolated nucleic acid can include a recombinant DNAmolecule that is part of a hybrid or fusion nucleic acid sequence.

In one example, the term “isolated” as used with reference to a nucleicacid molecule also includes any non-naturally-occurring nucleic acidmolecule since non-naturally-occurring nucleic acid sequences are notfound in nature and do not have immediately contiguous sequences in anaturally-occurring genome. For example, non-naturally-occurring nucleicacid molecules such as an engineered nucleic acid molecule is consideredto be an isolated nucleic acid molecule. Engineered nucleic acidmolecules can be made using common molecular cloning or chemical nucleicacid synthesis techniques. Isolated non-naturally-occurring nucleic acidmolecule can be independent of other sequences, or incorporated into avector, an autonomously replicating plasmid, a virus (such as aretrovirus, adenovirus, or herpes virus), or the genomic DNA of aprokaryote or eukaryote. In addition, a non-naturally-occurring nucleicacid molecule can include a nucleic acid molecule that is part of ahybrid or fusion nucleic acid sequence.

Nucleic acid molecule: Encompasses both RNA and DNA including, withoutlimitation, cDNA, genomic DNA, and synthetic (such as chemicallysynthesized) DNA. The nucleic acid molecule can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid molecule can bethe sense strand or the antisense strand. In addition, nucleic acidmolecule can be circular or linear.

Oligonucleotide: A linear polynucleotide (such as DNA or RNA) sequenceof at least 9 nucleotides, for example at least 12, 15, 18, 20, 25, 30,50, 100 or even 200 nucleotides long. In one example, an oligonucleotideis no more than 100 nucleotides in length, such as no more than 50nucleotides, such as no more than 25 nucleotides.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame. Configurations of separate genes that are transcribed intandem as a single messenger RNA are denoted as operons. Thus placinggenes in close proximity, for example in a plasmid vector, under thetranscriptional regulation of a single promoter, constitutes a syntheticoperon.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Probes and primers: A “probe” includes an isolated nucleic acid moleculecontaining a detectable label or reporter molecule. Exemplary labelsinclude radioactive isotopes, ligands, chemiluminescent agents,fluorophores, and enzymes. Methods for labeling and guidance in thechoice of labels appropriate for various purposes are discussed in, forexample, Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989, and Ausubel et al. (ed.) Current Protocols inMolecular Biology, Greene Publishing and Wiley-Interscience, New York(with periodic updates), 1987.

“Primers” are typically nucleic acid molecules having ten or morenucleotides (such as nucleic acid molecules having between about 10nucleotides and about 100 nucleotides). A primer can be annealed to acomplementary target nucleic acid strand by nucleic acid hybridizationto form a hybrid between the primer and the target nucleic acid strand,and then extended along the target nucleic acid strand by, for example,a DNA polymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, for example, by the polymerase chain reaction(PCR) or other nucleic-acid amplification methods.

Methods for preparing and using probes and primers are described, forexample, in references such as Sambrook et al. (ed.), Molecular Cloning:A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989; Ausubel et al. (ed.), CurrentProtocols in Molecular Biology, Greene Publishing andWiley-Interscience, New York (with periodic updates), 1987; and Innis etal., PCR Protocols: A Guide to Methods and Applications, Academic Press:San Diego, 1990. PCR primer pairs can be derived from a known sequence,for example, by using computer programs intended for that purpose suchas Primer (Version 0.5, © 1991, Whitehead Institute for BiomedicalResearch, Cambridge, Mass.).

One of skill in the art will appreciate that the specificity of aparticular probe or primer increases with the length, but that a probeor primer can range in size from a full-length sequence to sequences asshort as five consecutive nucleotides. Thus, for example, a primer of 20consecutive nucleotides can anneal to a target with a higher specificitythan a corresponding primer of only 15 nucleotides. Thus, in order toobtain greater specificity, probes and primers can be selected thatinclude, for example, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500,or more consecutive nucleotides.

Promoter: An array of nucleic acid control sequences which directtranscription of a nucleic acid. A promoter includes necessary nucleicacid sequences near the start site of transcription, such as a TATAelement. A promoter also optionally includes distal enhancer orrepressor elements which can be located as much as several thousand basepairs from the start site of transcription.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide in is more enriched than thepeptide is in its environment within a cell, such that the peptide issubstantially separated from cellular components (nucleic acids, lipids,carbohydrates, and other polypeptides) that may accompany it. In anotherexample, a purified peptide preparation is one in which the peptide issubstantially-free from contaminants, such as those that might bepresent following chemical synthesis of the peptide.

In one example, a peptide is purified when at least about 50% by weightof a sample is composed of the peptide, for example when at least about60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% or more of a sample iscomposed of the peptide. Examples of methods that can be used to purifya peptide, include, but are not limited to the methods disclosed inSambrook et al. (Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y., 1989, Ch. 17). Protein purity can be determined by, forexample, polyacrylamide gel electrophoresis of a protein sample,followed by visualization of a single peptide band upon staining thepolyacrylamide gel; high-pressure liquid chromatography; sequencing; orother conventional methods.

Recombinant: A recombinant nucleic acid molecule is one that has asequence that is not naturally occurring, has a sequence that is made byan artificial combination of two otherwise separated segments ofsequence, or both. This artificial combination can be achieved, forexample, by chemical synthesis or by the artificial manipulation ofisolated segments of nucleic acid molecules, such as genetic engineeringtechniques. Recombinant is also used to describe nucleic acid moleculesthat have been artificially manipulated, but contain the same regulatorysequences and coding regions that are found in the organism from whichthe nucleic acid was isolated.

Sequence identity/similarity: The identity/similarity between two ormore nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Sequencesimilarity can be measured in terms of percentage similarity (whichtakes into account conservative amino acid substitutions); the higherthe percentage, the more similar the sequences are. Homologs ororthologs of nucleic acid or amino acid sequences possess a relativelyhigh degree of sequence identity/similarity when aligned using standardmethods. This homology is more significant when the orthologous proteinsor cDNAs are derived from species which are more closely related (suchas human and mouse sequences), compared to species more distantlyrelated (such as human and C. elegans sequences).

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation can be found at the NCBI web site.

BLASTN is used to compare nucleic acid sequences, while BLASTP is usedto compare amino acid sequences. To compare two nucleic acid sequences,the options can be set as follows: -i is set to a file containing thefirst nucleic acid sequence to be compared (such as C:\seq1.txt); -j isset to a file containing the second nucleic acid sequence to be compared(such as C:\seq2.txt); -p is set to blastn; -o is set to any desiredfile name (such as C:\output.txt); -q is set to -1; -r is set to 2; andall other options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two sequences: C:\Bl2seq -i c:\seq1.txt -jc:\seq2.txt -p blastn -o c:\output.txt -q -1 -r 2.

To compare two amino acid sequences, the options of Bl2seq can be set asfollows: -i is set to a file containing the first amino acid sequence tobe compared (such as C:\seq1.txt); -j is set to a file containing thesecond amino acid sequence to be compared (such as C:\seq2.txt); -p isset to blastp; -o is set to any desired file name (such asC:\output.txt); and all other options are left at their default setting.For example, the following command can be used to generate an outputfile containing a comparison between two amino acid sequences: C:\Bl2seq-i c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the twocompared sequences share homology, then the designated output file willpresent those regions of homology as aligned sequences. If the twocompared sequences do not share homology, then the designated outputfile will not present aligned sequences.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a nucleic acid sequence that has1166 matches when aligned with a test sequence having 1154 nucleotidesis 75.0 percent identical to the test sequence (i.e.,1166÷1554*100=75.0). The percent sequence identity value is rounded tothe nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 arerounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 arerounded up to 75.2. The length value will always be an integer. Inanother example, a target sequence containing a 20-nucleotide regionthat aligns with 20 consecutive nucleotides from an identified sequenceas follows contains a region that shares 75 percent sequence identity tothat identified sequence (that is, 15÷20*100=75).

For comparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). Homologs are typically characterizedby possession of at least 70% sequence identity counted over thefull-length alignment with an amino acid sequence using the NCBI BasicBlast 2.0, gapped blastp with databases such as the nr or swissprotdatabase. Queries searched with the blastn program are filtered withDUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70).Other programs use SEG. In addition, a manual alignment can beperformed. Proteins with even greater similarity will show increasingpercentage identities when assessed by this method, such as at leastabout 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

When aligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9;extension gap 1 penalties). Proteins with even greater similarity to thereference sequence will show increasing percentage identities whenassessed by this method, such as at least about 60%, 70%, 75%, 80%, 85%,90%, 95%, 98%, 99% sequence identity. When less than the entire sequenceis being compared for sequence identity, homologs will typically possessat least 75% sequence identity over short windows of 10-20 amino acids,and can possess sequence identities of at least 85%, 90%, 95% or 98%depending on their identity to the reference sequence. Methods fordetermining sequence identity over such short windows are described atthe NCBI web site.

One indication that two nucleic acid molecules are closely related isthat the two molecules hybridize to each other under stringentconditions, as described above. Nucleic acid sequences that do not showa high degree of identity may nevertheless encode identical or similar(conserved) amino acid sequences, due to the degeneracy of the geneticcode. Changes in a nucleic acid sequence can be made using thisdegeneracy to produce multiple nucleic acid molecules that all encodesubstantially the same protein. Such homologous nucleic acid sequencescan, for example, possess at least about 60%, 70%, 80%, 90%, 95%, 98%,or 99% sequence identity determined by this method. An alternative (andnot necessarily cumulative) indication that two nucleic acid sequencesare substantially identical is that the peptide which the first nucleicacid encodes is immunologically cross reactive with the peptide encodedby the second nucleic acid.

One of skill in the art will appreciate that the particular sequenceidentity ranges are provided for guidance only; it is possible thatstrongly significant homologs could be obtained that fall outside theranges provided.

Transformed cell: A cell into which a nucleic acid molecule has beenintroduced, for example by molecular biology techniques. Transformationencompasses all techniques by which a nucleic acid molecule can beintroduced into such a cell, including, but not limited to, transfectionwith viral vectors, conjugation, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Variants, fragments or fusion proteins: The enzymes disclosed hereinthat can be used to produce 3-HP and derivatives thereof, includevariants, fragments, and fusions thereof that retain essentially thesame activity as the native enzyme. DNA sequences which encode for anenzyme (for example SEQ ID NO: 17, 19, 21, 23, 25 or 27), or a fusion,fragment or variant thereof, can be engineered to allow the protein tobe expressed in a cell, such as a yeast, bacteria, insect, plant, orcell. To obtain expression, the DNA sequence can be altered and operablylinked to other regulatory sequences. The final product, which containsthe regulatory sequences and the protein, is a vector. This vector canbe introduced into any cell, such as a eukaryotic, bacteria, insect, orplant cell. Once inside the cell the vector allows the protein to beproduced.

A fusion protein including an enzyme, such as an enzyme used to produce3-HP (or variant, polymorphism, mutant, or fragment thereof), linked toother amino acid sequences that do not inhibit the desired activity ofthe enzyme, for example the ability to convert beta-alanine to 3-HP. Inone example, the other amino acid sequences are no more than about 10,12, 15, 20, 25, 30, or 50 amino acids in length.

One of ordinary skill in the art will appreciate that a DNA sequence canbe altered in numerous ways without affecting the biological activity ofthe encoded protein. For example, PCR can be used to produce variationsin the DNA sequence which encodes an enzyme. Such variants can bevariants optimized for codon preference in a host cell used to expressthe protein, or other sequence changes that facilitate expression.

Vector: A nucleic acid molecule as introduced into a cell, therebyproducing a transformed cell. A vector can include nucleic acidsequences that permit it to replicate in the cell, such as an origin ofreplication. In particular examples, a vector also includes one or moreselectable markers or other genetic elements. A vector can also benonreplicating, such that it is a means of introducing nucleic acidsequences that are integrated into the genome of the cell.

Cells to Produce 3-HP and 3-HP Derivatives

Transformed cells having beta-alanine/pyruvate aminotransferase activity(EC 2.6.1.18) are disclosed. Such cells can produce 3-HP frombeta-alanine and pyruvate via a malonate semialdehyde intermediate.Transformed cells including beta-alanine/pyruvate aminotransferaseactivity can be eukaryotic or prokaryotic. Examples of such cellsinclude, but are not limited to Lactobacillus, Lactococcus, Bacillus,Escherichia, Geobacillus, Corynebacterium, Clostridium, fungal, plant,and yeast cells. In one example, a plant cell is part of a plant, suchas a transgenic plant. Transformed cells can include at least oneexogenous nucleic acid molecule that encodes a beta-alanine/pyruvateaminotransferase (EC 2.6.1.18), for example a sequence including SEQ IDNO: 17 or 19, or variants, fragments, or fusions thereof that retain theability to convert beta-alanine to malonate semialdehyde. The disclosedtransformed cells can further include other exogenous nucleic acidmolecules that encode other enzymes, such as 3-hydroxypropionatedehydrogenase (EC 1.1.1.59), alanine 2,3-aminomutase, lipase or esterase(EC 3.1.1.-), aldehyde dehydrogenase (EC 1.2.1.-), alcohol dehydrogenase(EC 1.1.1.1), or combinations thereof.

Cells that include beta-alanine/pyruvate aminotransferase activity, aswell as additional enzyme activities, are disclosed. In one example,transformed cells having beta-alanine/pyruvate aminotransferase activityalso have dehydrogenase activity, such as an enzyme capable ofconverting malonate semialdehyde to 3-HP, for example3-hydroxypropionate dehydrogenase activity. One non-limiting example ofan enzyme having 3-hydroxypropionate dehydrogenase activity is an mmsBenzyme from P. aeruginosa (such as SEQ ID NO: 28 and variants, fusions,and fragments thereof that retain 3-hydroxypropionate dehydrogenaseactivity).

In another example, transformed cells having beta-alanine/pyruvateaminotransferase activity and 3-hydroxypropionate dehydrogenase activityalso have alanine 2,3-aminomutase activity. Non-limiting examples of anenzyme having alanine 2,3-aminomutase activity include SEQ ID NO: 22, 24and 26, and variants, fusions, and fragments thereof that retain alanine2,3-aminomutase activity. In a particular example, a transformed cellincludes beta-alanine/pyruvate aminotransferase activity,3-hydroxypropionate dehydrogenase activity, alanine 2,3-aminomutaseactivity, as well as lipase or esterase activity, or a combination ofboth lipase activity and esterase activity. Such transformed cells canbe used to produce an ester of 3-HP, such as methyl 3-hydroxypropionate,ethyl 3-hydroxypropionate, propyl 3-hydroxypropionate, butyl3-hydroxypropionate, or 2-ethylhexyl 3-hydroxypropionate.

In another example, transformed cells having beta-alanine/pyruvateaminotransferase activity also include aldehyde dehydrogenase activityand alcohol dehydrogenase activity. These cells can be used to produce1,3-propanediol. In some examples, such cells further include3-hydroxypropionate dehydrogenase activity, alanine 2,3-aminomutaseactivity, or a combination of both 3-hydroxypropionate dehydrogenaseactivity and alanine 2,3-aminomutase activity.

In a particular example, transformed cells having beta-alanine/pyruvateaminotransferase activity also include esterase activity. These cellscan be used to produce polymerized 3-HP. In some examples, such cellsfurther include 3-hydroxypropionate dehydrogenase activity, alanine2,3-aminomutase activity, or a combination of both 3-hydroxypropionatedehydrogenase activity and alanine 2,3-aminomutase activity.

Pathways for Producing 3-HP and Derivatives Thereof

Provided herein are methods for producing 3-HP from beta-alanine byusing a beta-alanine/pyruvate aminotransferase. Severalbeta-alanine/pyruvate aminotransferase sequences and cells havingbeta-alanine/pyruvate aminotransferase activity, are disclosed. Inaddition, methods and materials related to producing esters of 3-HP,polymers of 3-HP, and 1,3-propanediol are provided.

Specifically, the disclosure provides a novel route for producing 3-HPfrom beta-alanine using beta-alanine/pyruvate aminotransferase nucleicacids (such as SEQ ID NO: 17, 19 and variants, fragments and fusionsthereof), peptides (such as SEQ ID NO: 18, 20 and variants, fragmentsand fusions thereof), which is more efficient and allows for theproduction of greater amounts of 3-HP as well as derivatives thereofsuch as 1,3-propanediol, polymerized 3-HP, and esters of 3-HP.

Pathways of 3-HP and its Derivatives

As shown in FIG. 1, beta-alanine can be converted into malonatesemialdehyde through the use of a peptide having beta-alanine/pyruvateaminotransferase activity (E.C. 2.6.1.18). Exemplarybeta-alanine/pyruvate aminotransferases include, but are not limited tosequences including SEQ ID NOS: 18 and 20, or fragments, variants, orfusions thereof that retain beta-alanine/pyruvate aminotransferaseactivity. Beta-alanine can be produced from aspartic acid by aspartatedecarboxylase. Beta-alanine can also be produced from alpha-alanine byendogenous peptides in a host cell that converts alpha-alanine tobeta-alanine. Beta-alanine can also be produced from alanine byalanine-2,3-aminomutase which converts alpha-alanine to beta-alanine.For example, a cell transformed with recombinant alanine 2,3-aminomutasenucleic acid, such as a sequence including SEQ ID NO: 21, 23, or 25, orfragments, variants, or fusions thereof that retain alanine2,3-aminomutase activity, can be used to produce beta-alanine.

Malonate semialdehyde produced from beta-alanine can then be convertedinto 3-HP through the use of a 3-HP dehydrogenase, such as a3-hydroxypropionate dehydrogenase. Exemplary 3-hydroxypropionatedehydrogenases include, but are not limited to sequences including SEQID NO: 28, or fragments, variants, or fusions thereof that retain3-hydroxypropionate dehydrogenase activity.

Derivatives of 3-HP can be made from beta-alanine as shown in FIG. 1.For example, the resulting 3-HP can be converted into an ester of 3-HPby a peptide having lipase or esterase activity, or a combinationthereof (EC 3.1.1.-). In addition, the resulting 3-HP can be convertedinto a polymerized 3-HP by a peptide having esterase activity.

The resulting 3-HP can also be converted into 1,3-propanediol by apeptide having aldehyde dehydrogenase activity (EC 1.2.1.-) and apeptide having alcohol dehydrogenase activity (EC 1.1.1.1). In addition,1,3-propanediol can be generated using peptides having oxidoreductaseactivity (such as enzymes in the 1.1.1.-class of enzymes) in vitro or invivo. The formation of 1,3-propanediol during fermentation or in an invitro assay can be analyzed using a High Performance LiquidChromatography (HPLC). The chromatographic separation can be achieved byusing a Bio-Rad 87H ion-exchange column. A mobile phase of 0.01Nsulfuric acid is passed at a flow rate of 0.6 ml/min and the columnmaintained at a temperature of 45-65° C. The presence of 1,3-propanediolin the sample can be detected using a refractive index detector (Skralyet al., Appl. Environ. Microbiol. 64:98-105, 1998).

In another example, 3-HP can be dehydrated to form acrylic acid. Anymethod can be used to perform a dehydration reaction. For example, 3-HPcan be heated in the presence of a catalyst (such as a metal or mineralacid catalyst) to form acrylic acid.

Enzymes

Peptides having beta-alanine/pyruvate aminotransferase activity, as wellas nucleic acid molecules encoding such peptides, can be obtained fromvarious species including, but not limited to: Pseudomonas putida,Pseudomonas aeruginosa, Arabidopsis thaliana, Rattus norvegicus, andXenopus laevis. For example, nucleic acid sequences havingbeta-alanine/pyruvate aminotransferase are shown in SEQ ID NO: 17 forPseudomonas putida (the corresponding amino acid sequence is shown inSEQ ID NO: 18), and in SEQ ID NO: 19 for Pseudomonas aeruginosa (thecorresponding amino acid sequence is shown in SEQ ID NO: 20). Inaddition, other peptides having beta-alanine/pyruvate aminotransferaseas well as nucleic acid molecules encoding such peptides, can beobtained using the methods described herein. For example,beta-alanine/pyruvate aminotransferase variants havingbeta-alanine/pyruvate aminotransferase activity can be used.

Peptides having alanine 2,3-aminomutase activity, as well as nucleicacid molecules encoding such peptides, can be obtained from variousspecies including, but not limited to: Bacillus subtilis, Porphyromonasgingivalis, Clostridium sticklandii, or Fusobacterium nucleatum. Forexample, nucleic acid sequences having alanine 2,3-aminomutase activityare shown in SEQ ID NO: 21 and 23 for B. subtilis (the correspondingamino acid sequences are shown in SEQ ID NO: 22 and 24, respectively),and in SEQ ID NO: 25 for P. gingivalis (the corresponding amino acidsequence is shown in SEQ ID NO: 26). Alanine 2,3-aminomutase variantshaving alanine 2,3-aminomutase activity can also be used.

Peptides having 3-hydroxypropionate dehydrogenase activity (EC1.1.1.59), such as that encoded by the mmsB gene of Pseudomonasaeruginosa, as well as nucleic acid molecules encoding such peptides,can be obtained from various species. For example, a nucleic acidmolecule that encodes a peptide having 3-hydroxypropionate dehydrogenaseactivity is shown in SEQ ID NO: 27 (see also GenBank Accession No:AE004778 (for protein, see GenBank Accession No: AAG06957) and GenBankAccession No: M84911 (for protein, see GenBank Accession No: AAA25891)).

Peptides having lipase or esterase activity as well as nucleic acidmolecules encoding such peptides can be obtained from various speciesincluding, without limitation, Candida rugosa, Candida tropicalis, andCandida albicans. For example, nucleic acid molecules that encode apeptide having lipase activity can be obtained from C. rugosa and canhave a sequence as set forth in GenBank accession number A81171 (PCTPublication No. WO/9914338).

Peptides having aldehyde dehydrogenase (NAD(P)+) (EC 1.2.1.-) activityas well as nucleic acid molecules encoding such peptides can be obtainedfrom various species including, without limitation, S. cerevisiae. Forexample, nucleic acid molecules that encode a peptide having aldehydedehydrogenase activity can be obtained from S. cerevisiae and can have asequence as set forth in GenBank Accession No. Z75282 (Tessier et al.FEMS Microbiol. Lett. 164:29-34, 1998).

Peptides having alcohol dehydrogenase activity (EC 1.1.1.1) as well asnucleic acid molecules encoding such peptides can be obtained fromvarious species including, without limitation, Z. mobilis. For example,a nucleic acid molecule that encodes a peptide having alcoholdehydrogenase activity can be obtained from Z. mobilis and can have asequence as set forth in GenBank accession No. M32100.

The term “peptide having enzymatic activity” refers to any peptide thatcatalyzes a chemical reaction of other substances without itself beingdestroyed or altered upon completion of the reaction. Typically, apeptide having enzymatic activity catalyzes the formation of one or moreproducts from one or more substrates. Such peptides can have any type ofenzymatic activity including, without limitation, the enzymatic activityor enzymatic activities associated with enzymes such asbeta-alanine/pyruvate aminotransferase, dehydrogenases capable ofconverting malonate semialdehyde to 3-HP such as 3-hydroxypropionatedehydrogenase, alanine 2,3-aminomutase, lipase or esterase, aldehydedehydrogenase, and alcohol dehydrogenase.

Methods of Making 3-HP and Derivatives Thereof

Each step provided in the pathways depicted in FIG. 1 can be performedwithin a cell (in vivo) or outside a cell (in vitro, for example in acontainer or column). Additionally, the organic compound products can begenerated through a combination of in vivo synthesis and in vitrosynthesis. Moreover, the in vitro synthesis step, or steps, can be viachemical reaction or enzymatic reaction.

For example, a cell or microorganism provided herein can be used toperform the steps provided in FIG. 1, or an extract containing peptideshaving the indicated enzymatic activities can be used to perform thesteps provided in FIG. 1. In addition, chemical treatments can be usedto perform the conversions provided in FIG. 1.

Expression of Peptides

The peptides described herein, such as the enzymes listed in FIG. 1, canbe produced individually in a host cell or in combination in a hostcell. Moreover, the peptides having a particular enzymatic activity canbe a peptide that is either naturally-occurring ornon-naturally-occurring. A naturally-occurring peptide is any peptidehaving an amino acid sequence as found in nature, including wild-typeand polymorphic peptides. Naturally-occurring peptides can be obtainedfrom any species including, but not limited to, animal (such asmammalian), plant, fungal, and bacterial species. Anon-naturally-occurring peptide is any peptide having an amino acidsequence that is not found in nature. Thus, a non-naturally-occurringpeptide can be a mutated version of a naturally-occurring peptide, or anengineered peptide. A peptide can be mutated by, for example, sequenceadditions, deletions, substitutions, or combinations thereof.

Genetically modified cells are disclosed which can be used to performone or more steps of the steps in the pathways described herein or thegenetically modified cells can be used to produce the disclosed peptidesfor subsequent use in vitro. For example, an individual microorganismcan contain one or more exogenous nucleic acids encoding each peptide toperform the steps depicted in FIG. 1. Such cells can contain any numberof exogenous nucleic acid molecules. For example, a particular cell cancontain one, two, three, four, five, six, or seven, or even moredifferent exogenous nucleic acid molecules with each one encoding apeptide for converting beta-alanine into 3-HP as shown in FIG. 1, or aparticular cell can endogenously produce peptides for convertingmalonate semialdehyde into 3-HP while containing an exogenous nucleicacid molecule that encodes peptides for converting beta-alanine intomalonate semialdehyde and in some examples an exogenous nucleic acidmolecule that encodes peptides that can convert alpha-alanine intobeta-alanine.

In addition, a single exogenous nucleic acid molecule can encode one, ormore than one, peptide. For example, a single exogenous nucleic acidmolecule can include sequences that encode two, three, four, five, six,seven, or even more different peptides. Further, the cells describedherein can contain a single copy, or multiple copies (such as about 5,10, 20, 35, 50, 75, 100 or 150 copies), of a particular exogenousnucleic acid molecule, such as a particular enzyme. The cells describedherein can contain more than one particular exogenous nucleic acidsequence. For example, a particular cell can contain about 50 copies ofexogenous nucleic acid molecule X as well as about 75 copies ofexogenous nucleic acid molecule Y.

In one example, a cell includes an exogenous nucleic acid molecule thatencodes a peptide having BAPAT activity, for example SEQ ID NO: 17 or 19(or variants, fragments, or fusions thereof that retain BAPAT activity).Such cells can have any detectable level of BAPAT activity, includingactivity detected by the production of metabolites of beta-alanine. Forexample, a cell containing an exogenous nucleic acid molecule thatencodes a peptide having BAPAT activity can have BAPAT activity with aspecific activity greater than about 1 μg malonate semialdehyde formedper gram dry cell weight per hour (for example greater than about 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, 400,500, or more μg malonate semialdehyde formed per gram dry cell weightper hour). Alternatively, a cell can have BAPAT activity such that acell extract from 1×10⁶ cells has a specific activity greater than about1 ng malonate semialdehyde formed per mg total protein per minute (suchas greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150,200, 250, 300, 350, 400, 500, or more ng 3-HP formed per mg totalprotein per minute).

Host Cells for Production of 3-HP and Related Products

The nucleic acid and amino acid sequences provided herein can be usedwith cells to produce 3-HP as well as derivatives thereof such as estersof 3-HP, polymerized 3-HP and 1,3-propanediol, for example using thepathways shown in Example 1. Such cells can be from any species, such asthose listed within the taxonomy web pages at the National Institutes ofHealth. The cells can be eukaryotic or prokaryotic. For example,genetically modified cells can be mammalian cells (such as human,murine, and bovine cells), plant cells (such as corn, wheat, rice, andsoybean cells), fungal cells (such as Aspergillus and Rhizopus cells),yeast cells, or bacterial cells (such as Lactobacillus, Lactococcus,Bacillus, Escherichia, and Clostridium cells).

In one example, a cell is a microorganism. The term “microorganism”refers to any microscopic organism including, but not limited to,bacteria, algae, fungi, and protozoa. Thus, E. coli, B. subtilis, B.licheniformis, S. cerevisiae, Kluveromyces lactis, Candida blankii,Candida rugosa, and Pichia pastoris are microorganisms and can be usedas described herein. In another example, the cell is part of a largerorganism, such as a plant, such as a transgenic plant. Examples ofplants that can be used to make 3-HP or other organic compounds frombeta-alanine include, but are not limited to, genetically engineeredplant crops such as corn, rice, wheat, and soybean.

In one example, cells that are genetically modified to synthesize aparticular organic compound contain one or more exogenous nucleic acidmolecules that encode peptides having specific enzymatic activities. Forexample, a microorganism can contain exogenous nucleic acid that encodesa peptide having BAPAT activity. In this case, beta-alanine can beconverted into malonate semialdehyde, which can lead to the productionof 3-HP. An exogenous nucleic acid molecule that encodes a peptidehaving an enzymatic activity that catalyzes the production of a compoundnot normally produced by a cell, can be introduced into the cell.Alternatively, a cell can be transformed with an exogenous nucleic acidmolecule that encodes a peptide having an enzymatic activity thatcatalyzes the production of a compound that is normally produced by thatcell. In this case, the genetically modified cell can produce more ofthe compound (such as 3-HP, derivatives thereof, or a combination of3-HP and its derivatives), or can produce the compound more efficiently,than a similar cell not having the genetic modification.

The produced one or more products can be secreted from the cell,eliminating the need to disrupt cell membranes to retrieve the organiccompound. In one example, the cell produces 3-HP, derivatives thereof,or both, with the concentration of the one or more products being atleast about 1 mg per L (such as at least about 1 mg/L, 5 mg/L, 10 mg/L,25 mg/L, −100 mg/L, 500 mg/L, 1 g/L, 5 g/L, 10 g/L, 25 g/L, 50 g/L, 100g/L or 150 g/L). When determining the yield of a compound such as 3-HPor derivatives thereof for a particular cell, any method can be used(such as Applied Environmental Microbiology 59(12):4261-5, 1993). A cellwithin the scope of the disclosure can utilize a variety of carbonsources.

A cell can contain one or more exogenous nucleic acid molecules thatencodes one or more peptides having enzymatic activity that leads to theformation of 3-HP or derivatives thereof, such as 1,3-propanediol,3-HP-esters, and polymers and copolymers containing 3-HP. Methods ofidentifying cells that contain exogenous nucleic acid molecules are wellknown. Such methods include, without limitation, PCR and nucleic acidhybridization techniques such as Northern and Southern analysis (seehybridization described herein). In addition, immunohisto-chemical andbiochemical techniques can be used to determine if a cell contains aparticular nucleic acid sequence by detecting the expression of thepeptide encoded by that particular nucleic acid molecule. For example,an antibody having specificity for a peptide can be used to determinewhether or not a particular cell contains a nucleic acid moleculeencoding that peptide.

Biochemical techniques can also be used to determine if a cell containsa particular nucleic acid molecule encoding a peptide having enzymaticactivity by detecting an organic product produced as a result of theexpression of the peptide having enzymatic activity. For example,detection of 3-HP after introduction of one or more exogenous nucleicacid molecules that encode a peptide having BAPAT activity (and in someexamples also introducing an exogenous nucleic acid molecule encoding apeptide having 3-hydroxypropionate dehydrogenase activity) into a cellthat does not normally express such a peptide can indicate that the cellnot only contains the introduced exogenous nucleic acid molecule butalso expresses the encoded peptide from that introduced exogenousnucleic acid molecule.

Methods for detecting specific enzymatic activities or the presence ofparticular organic products are well known, for example, the presence ofan organic compound such as 3-HP can be determined as described inSullivan and Clarke (J. Assoc. Offic. Agr. Chemists, 38:514-8, 1955).

Operons for Producing 3-HP and Derivatives Thereof

Operons including more than one coding sequence are disclosed. In oneexample, the operon includes a nucleic acid sequence encoding abeta-alanine/pyruvate aminotransferase, and a nucleic acid sequenceencoding a dehydrogenase capable of converting malonate semialdehyde to3-HP, such as 3-hydroxypropionate dehydrogenase. In a particularexample, the operon further includes a nucleic acid sequence encoding analanine 2,3-aminomutase. In yet other examples, the operon furtherincludes a nucleic acid sequence encoding a lipase or esterase, orcombinations thereof, which can be used to produce 3-HP esters. Inanother example, the operon further includes a nucleic acid sequenceencoding an esterase which can be used to produce polymers of 3-HP. Instill other examples, the operon further includes a nucleic acidsequence encoding an alcohol dehydrogenase and an aldehyde dehydrogenasewhich can be used to produce 1,3-propanediol. These recombinant operonscan also include a promoter, to drive expression of the codingsequences. Furthermore, the operons can be part of a vector, which isused to transform a cell that can be used to produce 3-HP or derivativesthereof.

Nucleic Acids and Proteins for Producing 3-HP and Derivatives Thereof

Enzymes that can be used to produce 3-HP and derivatives thereof aredisclosed herein. Several exemplary enzyme sequences are disclosedherein; however, the disclosure also encompasses variants, fusions, andfragments of the enzymes that retain the particular enzyme activity.

The nucleic acid sequences encoding the enzymes disclosed herein cancontain an entire nucleic acid sequence encoding the enzyme, as well asportions thereof that retain the desired enzyme activity. For example,an enzyme nucleic acid sequence can contain at least about 12 contiguousnucleotides of an enzyme nucleic acid sequence. It will be appreciatedthat the disclosure also provides isolated nucleic acid that contains anucleotide sequence that is greater than about 12 nucleotides (such asat least 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 100, 250,500, 750, 1000, 1500, 2000, 3000, 4000, or 5000 bases) in length andidentical to any portion of an enzyme sequence. The disclosure alsoprovides isolated nucleic acid sequences that encode for an enzymehaving at least about 12 bases and hybridizes, under moderately orhighly stringent hybridization conditions, to the sense or antisensestrand of a nucleic acid sequence encoding the desired enzyme.

In addition, the disclosure provides isolated enzyme nucleic acidsequences which contain a variation of an enzyme sequence. Variants cancontain a single insertion, a single deletion, a single substitution,multiple insertions, multiple deletions, multiple substitutions, or anycombination thereof (such as a single deletion together with multipleinsertions) as long as the peptide encoded thereby retains theappropriate activity. Such isolated nucleic acid molecules can share atleast about 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%sequence identity with an enzyme sequence, as long as the peptideencoded by the nucleic acid sequence retains the desired enzymeactivity. For example, the following variations can be made to analanine 2,3-aminomutase nucleic acid sequence: for SEQ ID NO: 21 or 23,the “a” at position 12 can be substituted with an “g”; the “g” atposition 1050 can be substituted with an “a”; the “a” at position 255;can be substituted with an “g” “t” or “c;” for SEQ ID NO: 25, the “a” atposition 6 can be substituted with a “g” “t” or “c”; the “t” at position66 can be substituted with a “c”; and the “g” at position 315; can besubstituted with an “a” “t” or “c.” Similarly, the following variationscan be made to a beta-alanine/pyruvate aminotransferase nucleic acidsequence: for SEQ ID NO: 17, the “c” at position 12 can be substitutedwith a “g” “t” or “a”; the “t” at position 336 can be substituted withan “c”; the “g” at position 1104 can be substituted with an “a” “t” or“c;” for SEQ ID NO: 19, the “g” at positions 51 and 528 can besubstituted with a “a” “t” or “c”; the “c” at position 672 can besubstituted with a “t” or an “a”; and the “g” at position 1296; can besubstituted with an “a”.

Codon preferences and codon usage tables for a particular species can beused to engineer isolated nucleic acid molecules that take advantage ofthe codon usage preferences of that particular species. For example, theenzymes disclosed herein can be designed to have codons that arepreferentially used by a particular organism of interest.

Nucleic acid molecules encoding a peptide can be produced by standardDNA mutagenesis techniques, for example, M13 primer mutagenesis. Detailsof these techniques are provided in Sambrook et al. (ed.), MolecularCloning: A Laboratory Manual 2nd ed., vol. 1-3, Cold Spring HarborLaboratory Press, Cold Spring, Harbor, N.Y., 1989, Ch. 15. Nucleic acidmolecules can contain changes of a coding region to fit the codon usagebias of the particular organism into which the molecule is to beintroduced.

Alternatively, the coding region can be altered by taking advantage ofthe degeneracy of the genetic code to alter the coding sequence in sucha way that, while the nucleic acid sequence is substantially altered, itnevertheless encodes a peptide having an amino acid sequence identicalor substantially similar to the native amino acid sequence. For example,because of the degeneracy of the genetic code, alanine is encoded by thefour nucleotide codon triplets: GCT, GCA, GCC, and GCG. Thus, thenucleic acid sequence of the open reading frame can be changed at analanine position to any of these codons without affecting the amino acidsequence of the encoded peptide or the characteristics of the peptide.Based upon the degeneracy of the genetic code, nucleic acid variants canbe derived from a nucleic acid sequence using standard DNA mutagenesistechniques as described herein, or by synthesis of nucleic acidsequences. Thus, this disclosure also encompasses nucleic acid moleculesthat encode the same peptide but vary in nucleic acid sequence by virtueof the degeneracy of the genetic code.

Identification of Alternative Sequences

A nucleic acid molecule encoding a peptide having the desired enzymaticactivity can be identified and obtained using any method such as thosedescribed herein. For example, nucleic acid molecules that encode apeptide having the desired enzymatic activity can be identified andobtained using common molecular cloning or chemical nucleic acidsynthesis procedures and techniques, including PCR. In addition,standard nucleic acid sequencing techniques and software programs thattranslate nucleic acid sequences into amino acid sequences based on thegenetic code can be used to determine whether or not a particularnucleic acid has any sequence homology with known enzymatic peptides.Sequence alignment software such as MEGALIGN (DNASTAR, Madison, Wis.,1997) can be used to compare various sequences.

In addition, nucleic acid molecules encoding known enzymatic peptidescan be altered using common molecular cloning techniques (such assite-directed mutagenesis). Possible alterations include, withoutlimitation, deletions, insertions, and substitutions, and combinationsthereof. Further, nucleic acid and amino acid databases (such as GenBankand EMBL-EBI) can be used to identify a nucleic acid sequence thatencodes a peptide having the desired enzymatic activity. Briefly, anyamino acid sequence having some homology to a peptide having enzymaticactivity, or any nucleic acid sequence having some homology to asequence encoding a peptide having enzymatic activity can be used as aquery to search GenBank. The identified peptides then can be analyzed todetermine whether or not they exhibit enzymatic activity.

In addition, nucleic acid hybridization techniques can be used toidentify and obtain a nucleic acid molecule that encodes a peptidehaving enzymatic activity. Briefly, any nucleic acid molecule thatencodes a known enzymatic peptide, or fragment thereof, can be used as aprobe to identify similar nucleic acid molecules by hybridization underconditions of moderate to high stringency. Such similar nucleic acidmolecules then can be isolated, sequenced, and analyzed to determinewhether the encoded peptide has similar enzymatic activity.

Expression cloning techniques also can be used to identify and obtain anucleic acid molecule that encodes a peptide having the desiredenzymatic activity. For example, a substrate known to interact with aparticular enzymatic peptide can be used to screen a phage displaylibrary containing that enzymatic peptide. Phage display libraries canbe generated as described (Burritt et al., Anal. Biochem. 238:1-13,1990), or can be obtained from commercial suppliers such as Novagen(Madison, Wis.).

Further, peptide sequencing techniques can be used to identify andobtain a nucleic acid molecule that encodes a peptide having the desiredenzymatic activity. For example, a purified peptide can be separated bygel electrophoresis, and its amino acid sequence determined by, forexample, amino acid microsequencing techniques. Once determined, theamino acid sequence can be used to design degenerate oligonucleotideprimers. Degenerate oligonucleotide primers can be used to obtain thenucleic acid sequence encoding the peptide by PCR. Once obtained, thenucleic acid molecule can be sequenced, cloned into an appropriateexpression vector, and introduced into a microorganism.

Transforming Cells

Any method can be used to introduce an exogenous nucleic acid moleculeinto a cell. For example, heat shock, lipofection, electroporation,conjugation, fusion of protoplasts, and biolistic delivery are commonmethods for introducing nucleic acid into bacteria and yeast cells. (forexample, see Ito et al., J. Bacterol. 153:163-8, 1983; Durrens et al.,Curr. Genet. 18:7-12, 1990; Sambrook et al., Molecular cloning: Alaboratory manual, Cold Spring Harbour Laboratory Press, New York, USA,second edition, 1989; and Becker and Guarente, Methods in Enzymology194:182-7, 1991). Other methods for expressing an amino acid sequencefrom an exogenous nucleic acid molecule include, but are not limited to,constructing a nucleic acid molecule such that a regulatory elementpromotes the expression of a nucleic acid sequence that encodes apeptide. Typically, regulatory elements are DNA sequences that regulatethe expression of other DNA sequences at the level of transcription.Thus, regulatory elements include, without limitation, promoters,enhancers, and the like.

Any type of promoter can be used to express an amino acid sequence froman exogenous nucleic acid molecule. Examples of promoters include,without limitation, constitutive promoters, tissue-specific promoters,and promoters responsive or unresponsive to a particular stimulus (suchas light, oxygen, chemical concentration). Methods for transferringnucleic acids into mammalian cells are also known, such as using viralvectors.

An exogenous nucleic acid molecule contained within a particular cell ofthe disclosure can be maintained within that cell in any form. Forexample, exogenous nucleic acid molecules can be integrated into thegenome of the cell or maintained in an episomal state. That is, a cellcan be a stable or transient transformant. A microorganism can containsingle or multiple copies (such as about 5, 10, 20, 35, 50, 75, 100 or150 copies), of a particular exogenous nucleic acid molecule, such as anucleic acid encoding an enzyme.

In Vitro Production of Organic Acids and Related Products

Purified peptides having enzymatic activity can be used alone, or incombination with cells, to produce 3-HP, derivatives thereof, or both,such as esters of 3-HP, polymerized 3-HP, and 1,3-propanediol. Further,cell-free extracts containing a peptide having enzymatic activity can beused alone or in combination with purified peptides, cells, or both, toproduce 3-HP, derivatives thereof, or both.

For example, a cell-free extract that includes a peptide having BAPATactivity can be used to form malonate semialdehyde from beta-alanine,while a cell or microorganism containing peptides having the enzymaticactivities necessary to catalyze the reactions needed to form 3-HP frommalonate semialdehyde can be used to produce 3-HP. In another example, acell-free extract which includes an alanine 2,3-aminomutase can be usedto form beta-alanine from alpha-alanine. Any method can be used toproduce a cell-free extract. For example, osmotic shock, sonication, ora repeated freeze-thaw cycle followed by filtration or centrifugationcan be used to produce a cell-free extract from intact cells.

A cell, purified peptide, cell-free extract, or combinations thereof canbe used to produce 3-HP that is, in turn, treated chemically to produceanother compound. For example, a cell or microorganism can be used toproduce 3-HP, while a chemical process is used to modify 3-HP into aderivative such as polymerized 3-HP or an ester of 3-HP. Likewise, achemical process can be used to produce a particular compound that is,in turn, converted into 3-HP or other organic compound (such as estersof 3-HP, and polymerized 3-HP) using a cell, substantially pure peptide,or cell-free extract described herein.

Fermentation of Cells to Produce Organic Acids

A method is provided for producing 3-HP, derivatives thereof, or both,by culturing production cells, such as a microorganism, in culturemedium such that 3-HP, derivatives of 3-HP, or both, are produced. Ingeneral, the culture media and culture conditions can be such that thecells grow to an adequate density and produce the product efficiently.For large-scale production processes, any method can be used such asthose described elsewhere (Manual of Industrial Microbiology andBiotechnology, 2^(nd) Edition, Editors: Demain and Davies, ASM Press;and Principles of Fermentation Technology, Stanbury and Whitaker,Pergamon).

Briefly, a large tank (such as a 100 gallon, 200 gallon, 500 gallon, ormore tank) containing appropriate culture medium with, for example, aglucose carbon source is inoculated with a particular cell ormicroorganism. After inoculation, the cells or microorganisms areincubated to allow biomass to be produced. Once a desired biomass isreached, the broth containing the cells or microorganisms can betransferred to a second tank. This second tank can be any size. Forexample, the second tank can be larger, smaller, or the same size as thefirst tank. Typically, the second tank is larger than the first suchthat additional culture medium can be added to the broth from the firsttank. In addition, the culture medium within this second tank can be thesame as, or different from, that used in the first tank. For example,the first tank can contain medium with xylose, while the second tankcontains medium with glucose.

Once transferred, the cells or microorganisms can be incubated to allowfor the production of 3-HP, derivatives thereof, or both. Once produced,any method can be used to isolate the formed product. For example,common separation techniques can be used to remove the biomass from thebroth, and common isolation procedures (such as extraction,distillation, and ion-exchange procedures) can be used to obtain the3-HP or derivatives thereof from the cell-free broth. Alternatively, theproduct can be isolated while it is being produced, or it can beisolated from the broth after the product production phase has beenterminated.

Example 1 Construction of E. coli Deletion Strains

This example describes methods used to generate E. coli hosts for thecloning and expression of beta-alanine/pyruvate aminotransferase andother genes. E. coli bacteria were obtained from commercial sourceswhere noted, or constructed by the gene insertional inactivation methodof Datsenko and Wanner (Proc. Natl. Acad. Sci. USA 97: 6640-5, 2000)using E. coli strains BW25113/pKD46 and BW 25141/pKD3 (E. Coli GeneticStock Center, New Haven, Conn.).

Lactate dehydrogenase catalyzes the formation of lactic acid. Deletionof this gene, and hence elimination of lactic acid formation, isadvantageous for the detection of the formation of 3-HP because of thesimilarity in structure and chromatographic behavior of these twocompounds. The ΔldhA::cam strain, which has an insertion of achloramphenicol resistance marker gene into the ldhA locus, wasconstructed as follows.

The CAT gene conferring chloramphenicol resistance of pKD3 from BW25141/pKD3 was PCR amplified using:5′-ATATTTTTAGTAGCTTAAATGTGATTCAACATCACTGGAGGTGTAGGCTGGAGC TGCTTC (SEQ IDNO: 1), and 5′-TATCTGAATCAGCTCCCCTGGAATGCAGGGGAGCGGCAAGCATATGAATATCCTCCTTAG (SEQ ID NO: 2), where the underlined portion corresponds to theregions in the E. coli chromosome immediately upstream and downstream ofthe ldhA locus, respectively, and the non-underlined regions arehomologous to regions in pKD3 that permit amplification of a fragmentcontaining the CAT gene.

The PCR reaction contained 30 μl 10× concentrated PCR buffer (RocheDiagnostics, Indianapolis Ind.), approximately 100 ng plasmid pKD3, 0.2mM each dNTP, 0.2 μM each SEQ ID NO: 1 and 2, and 15 units Taqpolymerase (Roche Diagnostics) in a final volume of 300 μl. The PCRreaction was incubated at 95° C. for 30 seconds followed by 30 cycles of95° C. for 30 seconds, 45° C. for 30 seconds, and 72° C. for 1 minute,then held at 72° C. for 10 minutes. The PCR product was precipitatedwith ethanol, digested with DpnI (New England Biolabs, Beverly Mass.)purified with the QIAquick PCR Purification Kit (Qiagen, ValenciaCalif.), and transformed into BW25113/pKD46 expressing the recombinationfunctions. Transformants were plated on LB plates containing 25 μg/mlchloramphenicol. Chloramphenicol-resistant transformations weresingle-colony purified on non-selective LB medium at 42° C., and singlecolonies tested for retention of chloramphenicol resistance and loss ofampicillin resistance (indicating curing of pKD46). Confirmation ofcorrect insertion of the CAT gene into the ldhA locus was performedusing colony PCR of the resultant ΔldhA::cam strain using primers thatflank the insertion locus:

5′-TTCAATATCGCCATAGCTTTCA; (SEQ ID NO: 3) and 5′-GAGGATGAAAGGTCATTGG.(SEQ ID NO: 4)

Whereas the wild-type ldhA locus is expected to yield a PCR product of1112 basepairs (bp) which does not possess a PvuII restrictionendonuclease site, the ΔldhA::cam construct yielded a 1161-basepairproduct which is digested by PvuII to yield fragments of 709 and 452basepairs, thus confirming the insertion of the CAT gene into the ldhAlocus and disruption of the ldhA gene. Electrocompetent cells of E. coliΔldhA::cam or of other E. coli strains were generated and transformedusing standard methods, for example as described in Sambrook et al.Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989.

Deletions in the panD, yea, panC, and gabT genes were generated usingsimilar methods. Where necessary, the inserted chloramphenicolresistance marker gene was removed by the activity of the FLPrecombinase encoded by plasmid pCP20, as described in Datsenko andWanner (Proc. Natl. Acad. Sci. USA 97: 6640-5, 2000). For example, aderivative of the ΔpanD::cam strain in which the insertedchloramphenicol resistance marker gene is removed was constructed; thegenetic alteration in this strain is referred to as ΔpanD. Deletionswere made separately in E. coli BW 25113 and combined by P1transduction. The strain carrying the combination of ΔpanD, ΔyeiA,ΔpanC, ΔyfdE, and Δ(ygaF-gabD-gabT) is referred to as Car24, and thestrain referred to as Car27 contains the deletions carried in Car24 plusΔldhA::cam.

Example 2 Cloning a Pseudomonas aeruginosa Beta-Alanine/PyruvateAminotransferase

The complete amino acid sequence of a protein with beta-alanine/pyruvateaminotransferase activity from Pseudomonas putida IFO 14796 is publiclyavailable (GenBank Accession No. P28269; Yonaha et al., J. Biol. Chem.267:12506-10, 1992). Waters and Venables (FEMS Microbiol. Letts.34:279-82, 1986) describe the presence of this activity in P. aeruginosaPAO1, but do not disclose the sequence. Using the BLAST program toidentify genes in the P. aeruginosa PAO1 genome that are homologous tothe P. putida beta-alanine/pyruvate aminotransferase sequence, GenBankAccession No. NP_(—)248822 was identified as a potentialbeta-alanine/pyruvate aminotransferase homolog. Primers were designed toamplify this gene based on the complete P. aeruginosa genome (GenBankAccession No. AE004091). The PCR primers used were:5′-AAGCCCGAGGATCGACATATGAACCAGCCGCTC (SEQ ID NO: 5) and5′-CCACCTGCACGGTGGGTACGGC (SEQ ID NO: 6).

The PCR reaction (100 μl total volume) contained 1 μg template DNA (P.aeruginosa PAO1-LAC genomic DNA; American Type Culture Collection,Manassas, Va.; catalog No. 47085D), 0.2 μM each primer (SEQ ID NOS: 5and 6), 10 μl 10× Taq polymerase buffer (Roche Diagnostics), 0.2 mM eachnucleotide triphosphate, and 5 units Taq DNA polymerase (RocheDiagnostics). The mixture was heated at 95° C. for 3 minutes, thensubjected to 30 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds,and 72° C. for 90 seconds, and then held at 72° C. for an additional 7minutes. The reaction was purified with the QIAquick PCR PurificationKit (Qiagen), ethanol-precipitated in the presence of 3 μl Pellet PaintCo-Precipitant (Novagen, Madison, Wis.), treated with NdeI and NotI (NewEngland Biolabs, Beverly, Mass.), purified, and ligated with the QuickLigation Kit (New England Biolabs) into pPRONde similarly treated withthese restriction endonucleases.

The NdeI recognition site (underlined in SEQ ID NO: 5) was generated atthe 5′ end of the P. aeruginosa beta-alanine/pyruvate aminotransferasegene by the primer of SEQ ID NO: 5, while the NotI site occurs naturallystarting 3 nucleotides past (3′ of) the termination codon of this gene.Plasmid pPRONde is a derivative of pPROLar.A122 (Clontech Laboratories,Palo Alto, Calif.) in which an NdeI site was constructed at theinitiator ATG codon by oligonucleotide-directed mutagenesis using theQuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla Calif.).Expression of beta-alanine/pyruvate aminotransferase in pPRONde isdriven by a hybrid lac/ara promoter. Ligations were transformed intoelectrocompetent E. coli and clones verified by sequencing. The plasmidcarrying the P. aeruginosa beta-alanine/pyruvate aminotransferase geneis referred to herein as pPRO-PaBAPAT.

The cloned P. aeruginosa beta-alanine/pyruvate aminotransferase DNA isshown in SEQ ID NO: 19, and the corresponding protein in SEQ ID NO: 20.

Example 3 Cloning a P. putida Beta-Alanine/Pyruvate Aminotransferase

While the genome sequence of P. putida IFO 14796 has not beendetermined, the genome sequence of P. putida KT2440, and that of itsbeta-alanine/pyruvate aminotransferase homolog, is available (GenbankAccession No. NC_(—)002947.3). PCR primers were designed to amplify thisgene from P. putida genomic DNA (American Type Culture Collection47054D) in a manner similar to that used in Example 2:5′-TCTTCCGAGGAACCGCATATGAACATGCCCGAAAC (SEQ ID NO: 7) and5′-GCATACGCCTGGCATTAATTAAGGAAAGATCAGTCGATCAG (SEQ ID NO: 8).

NdeI and PacI (underlined sequences in SEQ ID NOS: 7 and 8) were used toclone the PCR product into pPRONde as described in Example 2. Theplasmid carrying the P. putida beta-alanine/pyruvate aminotransferasegene is referred to as pPRO-PpBAPAT. The cloned P. putidabeta-alanine/pyruvate aminotransferase cDNA is shown in SEQ ID NO: 17,and the corresponding protein in SEQ ID NO: 18.

One skilled in the art will understand that similar methods can be usedto clone a beta-alanine/pyruvate aminotransferase from other organisms,such as Streptomyces coelicolor A3, Corynebacterium glutamicum ATCC13032, and rat.

Example 4 Formation of 3-HP from Beta-Alanine Using BAPAT

E. coli BW25113 ΔldhA::cam cells carrying pPRONde or pPRO-PaBAPAT(Example 2) were grown in defined minimal medium (Neidhardt et al., J.Bacteriol. 119:736-47, 1974) plus 0.4% (w/v) glucose, 20 μg/mlpantothenate, and 25 μg/ml kanamycin at 37° C. to mid-log phase, andexpression of beta-alanine/pyruvate aminotransferase induced with 1 mMIPTG. Beta-alanine (50 mM final concentration) was added 50 minutesfollowing induction, and the culture centrifuged after 4 hours toseparate the cells from the medium. Samples of the culture supernatant(1 ml) were adjusted to ˜pH 2 with 50 μL formic acid, filtered, and 3-HPquantitated by HPLC followed by detection by mass spectrometry.

Chromatography was performed on a Waters 2690 liquid chromatographysystem using a 2.1 mm×250 mm Phenomenex Aqua C₁₈ reversed-phasechromatography column with isocratic elution at 45° C. using 1%methanol:aqueous containing 0.1% (v/v) formic acid as the mobile phaseat a flow rate of 0.18 ml/min. Parameters for the Micromass ZQquadrupole mass spectrometer operating in negative electrosprayionization mode (−ESI) were set as the following; capillary: 2.0 kV;cone: 25 V; extractor: 4 V; RF lens: 1 V; source temperature: 120° C.;desolvation temperature: 380° C.; desolvation gas: 600 L/h; cone gas:Off; low mass resolution: 15.0; high mass resolution: 15.0; ion energy:0.2; multiplier: 650. A selected ion monitoring MS parameter was set upto allow selection of m/z 89, corresponding to the deprotonatedmolecular ion, [M-H]⁻, of 3-HP.

Cells carrying pPRONde did not produce 3-HP, whereas cells carryingpPRO-PaBAPAT produced 0.04 g/L 3-HP. Increasing the incubation time to20 hours resulted in the formation of 0.06 g/L 3-HP. No 3-HP wasproduced in the absence of added beta-alanine.

Cells carrying pPRO-PpBAPAT, under the same conditions as those carryingpPRO-PaBAPAT, formed 0.10 g/L 3-HP in 20 hours. Increasing theconcentration of supplied beta-alanine to 250 mM in the medium resultedin the formation of 0.24 g/L 3-HP in 20 hours.

Example 5 Synthetic Operon BAPAT-mmsB for 3-HP Production

This example describes methods used to produce an operon that includes abeta-alanine/pyruvate aminotransferase gene and a 3-hydroxyisobutyratedehydrogenase (mmsB) gene encoding an enzyme with 3-hydroxypropionatedehydrogenase activity. Operons are configurations of genes that aretranscribed in tandem as a single messenger RNA. Thus placing genes inclose proximity, for example in a plasmid vector, under thetranscriptional regulation of a single promoter, constitutes a syntheticoperon.

Biosynthetic pathways that allow production of 3-HP via beta-alanine andpyruvate were generated (FIG. 1). One pathway to 3-HP from beta-alanineinvolves the use of a peptide having beta-alanine/pyruvateaminotransferase activity (EC 2.6.1.18), that is, an enzyme from a classof enzymes that convert beta-alanine to malonate semialdehyde, inaddition to an enzyme having 3-HP dehydrogenase activity (E.C.1.1.1.59), such as that encoded by a mmsB gene (such as SEQ ID NO: 27).

A synthetic operon that includes a beta-alanine/pyruvateaminotransferase gene and a mmsB gene was generated as follows. A mmsBgene was isolated from P. aeruginosa 633 genomic DNA (American TypeCulture Collection, 17933D) by PCR with primers:5′-ATACATATGACCGACATCGCATTCCTC (SEQ ID NO: 9) and5′-ATAGTCGACTTAGGGATGAAGCAGTGAG (SEQ ID NO: 10).

The DNA polymerase used was a mixture of rTth (1 U; Applied Biosystems)and Pfu (0.125 U, Stratagene, La Jolla Calif.), with the following PCRamplification program: 94° C. for 5 minutes, 25 cycles of 94° C. for 30seconds, 45° C. (increasing 0.3° C. per cycle) for 30 seconds, and 72°C. for 1 minute (increasing 2 seconds per cycle), followed by 72° C. for7 minutes. The PCR product was purified using the QIAquick PCRpurification kit (Qiagen) then digested with NdeI and SalI (New EnglandBiolabs). The resulting ˜1 kb fragment was gel purified using theQIAquick gel purification kit (Qiagen), and ligated into pET28a(Novagen, Madison Wis.) that had been similarly digested and purified.The ligation reaction was done with the Rapid Ligation kit (Roche.Applied Science), and transformed into TOP10 competent cells(Invitrogen, Carlsbad Calif.). The resultant plasmid is denotedpET28-mmsB.

The mmsB gene was amplified from pET28-mmsB using primers:5′-CAACGGCATCGCCTAATGAACGGCCGCTTAATTAAGAAGGAGGTASTAAATATG ACCGACATCG(SEQ ID NO: 11) and 5′-TTCGTTTTATTTGATGCCTCTAGATTAGTCCTTGCCGCGGTAGAGC(SEQ ID NO: 12).

The DNA polymerase used was cloned Pfu Turbo DNA pol (Stratagene) withthe following PCR amplification program: 95° C. for 30 seconds, 30cycles of 95° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 3minutes, followed by 72° C. for 10 minutes. The PCR reaction wasdigested with DpnI and the product gel-purified as above. The fragmentwas then used as a “mega-primer” in a QuikChange (Stratagene) extensionreaction as described by Kirsch and Joly (Nucl. Acids Res. 26:1848-50,1998), using pPRO-PaBAPAT as template. The resultant plasmid carrying aP. aeruginosa beta-alanine/pyruvate aminotransferase and an mmsB gene intandem under the expression control of the P_(lac/ara) promoter isreferred to as pBm1. The cloned mmsB cDNA sequence is shown in SEQ IDNO: 27, and the corresponding amino acid sequence in SEQ ID NO: 28.

Cells carrying pBm1 formed 0.09 g/L 3-HP when grown, expressed, and theculture medium analyzed as described in Example 4. These cells weredeposited with the American Type Culture Collection (Manassas, Va.) onDec. 6, 2004 (Accession No. PTA-6411).

Example 6 Synthetic Operon aam-BAPAT-mmsB for 3-HP Production

This example describes methods used to produce an operon that includes abeta-alanine/pyruvate aminotransferase gene, an mmsB gene, as well as analanine 2,3-aminomutase (aam) gene.

As shown in FIG. 1, beta-alanine can be produced from alpha-alanine byusing an alanine 2,3-aminomutase. The alanine 2,3-aminomutase gene usedwas derived from a Bacillus subtilis lysine 2,3-aminomutase bymutagenesis and selection (see WO 03/062173), and included in addition achange of tyrosine at amino acid position 140 to histidine (Y140H)(Bsaam2co, SEQ ID NO. 21). However, one skilled in the art willrecognize that other alanine 2,3-aminomutase genes can be used. Forexample, alternative alanine 2,3-aminomutase gene sequences include, butare not limited to, SEQ ID NOS: 23 and 25 as well as variants, fragmentsand fusions thereof that retain alanine 2,3-aminomutase activity.

The Bsaam2co alanine 2,3-aminomutase gene carried on a pPROLar-basedplasmid was amplified using: 5′-GAGCAATCACCTATGAACTG (SEQ ID NO: 13) and5′-GAGCGGCTGGTTCATTTGTACCTTCCTCCTCTTTAATGGCGGCCGCACCATTCGCATGTTTTTATGAAGAATCCC (SEQ ID NO: 14).

The DNA polymerase used was cloned Pfu Turbo DNA pol, with the followingPCR amplification program: 95° C. for 30 seconds, 30 cycles of 95° C.for 30 seconds, 54° C. for 30 seconds, and 72° C. for 4 minutes,followed by 72° C. for 20 minutes. The PCR reaction was digested withDpnI and the product gel-purified as above. The fragment was then usedas “mega-primer” in a QuikChange extension reaction as described above,using pBm1 as template. The resultant plasmid carrying the alanine2,3-aminomutase, the P. aeruginosa beta-alanine/pyruvateaminotransferase, and the mmsB genes (see Example 5) in tandem under theexpression control of the P_(lac/ara) promoter is referred to as pABm1.

A second operon including an alanine 2,3-aminomutase, a P. putidabeta-alanine/pyruvate aminotransferase (see Example 3), and an mmsB genein tandem under the expression control of the P_(lac/ara) promoter wasconstructed. The P. putida BAPAT gene was amplified from pPRO-PpBAPATusing primers: 5′-CACACAGAATGCGGCCGCGAGGAGAAAGGTAAATATGAACATGCCCG (SEQID NO: 15) and 5′-CGTTCACCGACAAACAACAG (SEQ ID NO: 16).

The DNA polymerase used was cloned Pfu Turbo DNA pol with the followingPCR amplification program: 95° C. for 2 minutes, 30 cycles of 95° C. for30 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes, followed by72° C. for 10 minutes. The PCR product was purified using the QIAquickPCR purification kit (Qiagen), digested with NotI and PacI (New EnglandBiolabs), the 1.4 kb fragment was gel purified and recovered using theQIAquick gel purification kit (Qiagen), and ligated into pABm1 similarlydigested and purified. The resultant plasmid carrying alanine2,3-aminomutase, P. putida beta-alanine/pyruvate aminotransferase, andmmsB in tandem under the expression control of the P_(lac/ara) promoteris referred to as pABm2.

Example 7 Expression, Purification, and Assay of P. putida BAPATActivity

The P. putida BAPAT cDNA cloned in Example 3 (SEQ ID NO: 17) wassubcloned from pPRO-PpBAPAT into pET-28b (Novagen, Madison Wis.) at theNdeI site to generate a protein with an additional 20 amino acids,including a His₆ purification tag, at the amino terminus. The subsequentplasmid, pET-PpBAPAT, was transformed into E. coli BL21(DE3) (Novagen)and protein production induced with 0.4 mM IPTG. Cells were disruptedwith Bugbuster (5 ml/g wet weight; Novagen) containing rLysozyme (3KU/ml; Novagen) and Benzonase (25 KU/ml; Novagen), and the cell-freeextract applied to His.Bind columns and the His6-BAPAT purifiedaccording to the manufacturer's instructions (Novagen).

The assay for BAPAT activity was performed by coupling the production ofmalonate semialdehyde to its reduction by a NADPH-dependent 3-HPdehydrogenase. The 3-HP dehydrogenase used was the product of the E.coli ydfG gene (Fujisawa et al., Biochim. Biophys. Acta 1645:89-94,2003). The assays were performed at 25° C. in 200 μl reactionscontaining 50 mM K phosphate buffer (pH 8.0), 0.1 mM pyridoxalphosphate, 20 mM β-alanine, 7.5 mM pyruvate, 1 mM NADPH, 0.5 Units of3-HP dehydrogenase, and purified His₆-BAPAT.

Enzymatic activity was monitored as the decrease in A₃₄₀ due to theoxidation of NADPH. The concentrations of β-alanine and pyruvate werevaried to determine the K_(m) for each substrate. Under theseconditions, the K_(m) for β-alanine was 2.2 mM, and that for pyruvatewas 1.2 mM. The specific activity of His₆-BAPAT was 3.9 Units/mg protein(1 Unit=1 μmol malonate semialdehyde produced per minute).

Example 8 Recombinant Expression

With publicly available enzyme cDNA and amino acid sequences, and theenzymes and sequences disclosed herein, such as beta-alanine/pyruvateaminotransferase, dehydrogenases such as 3-hydroxypropionatedehydrogenase, alanine 2,3-aminomutase, lipase or esterase, alcoholdehydrogenase, and aldehyde dehydrogenase, as well as variants,polymorphisms, mutants, fragments and fusions thereof, the expressionand purification of any protein by standard laboratory techniques isenabled. One skilled in the art will understand that enzymes andfragments thereof can be produced recombinantly in any cell or organismof interest, and purified prior to use, for example prior to productionof 3-HP and derivatives thereof.

Methods for producing recombinant proteins are well known in the art.Therefore, the scope of this disclosure includes recombinant expressionof any protein or fragment thereof, such as an enzyme. For example, seeU.S. Pat. No. 5,342,764 to Johnson et al.; U.S. Pat. No. 5,846,819 toPausch et al.; U.S. Pat. No. 5,876,969 to Fleer et al. and Sambrook etal. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,1989; Ch. 17).

Briefly, partial, full-length, or variant cDNA sequences that encode fora peptide can be ligated into an expression vector, such as a bacterialexpression vector. Proteins peptides can be produced by placing apromoter upstream of the cDNA sequence. Examples of promoters include,but are not limited to lac, trp, tac, trc, major operator and promoterregions of phage lambda, the control region of fd coat protein, theearly and late promoters of SV40, promoters derived from polyoma,adenovirus, retrovirus, baculovirus and simian virus, the promoter for3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, thepromoter of the yeast alpha-mating factors and combinations thereof.

Vectors suitable for the production of intact native proteins includepKC30 (Shimatake and Rosenberg, 1981, Nature 292:128), pKK177-3 (Amannand Brosius, 1985, Gene 40:183) and pET-3 (Studier and Moffatt, 1986, J.Mol. Biol. 189:113). A DNA sequence can be transferred to other cloningvehicles, such as other plasmids, bacteriophages, cosmids, animalviruses and yeast artificial chromosomes (YACs) (Burke et al., 1987,Science 236:806-12). These vectors can be introduced into a variety ofhosts including somatic cells, and simple or complex organisms, such asbacteria, fungi (Timberlake and Marshall, 1989, Science 244:1313-7),invertebrates, plants (Gasser and Fraley, 1989, Science 244:1293), andmammals (Pursel et al., 1989, Science 244:1281-8), which are renderedtransgenic by the introduction of the heterologous cDNA.

For expression in mammalian cells, a cDNA sequence can be ligated toheterologous promoters, such as the simian virus SV40, promoter in thepSV2 vector (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072-6), and introduced into cells, such as monkey COS-1 cells(Gluzman, 1981, Cell 23:175-82), to achieve transient or long-termexpression. The stable integration of the chimeric gene construct may bemaintained in mammalian cells by biochemical selection, such as neomycin(Southern and Berg, 1982, J. Mol. Appl. Genet. 1:327-41) andmycophoenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072-6).

The transfer of DNA into eukaryotic, such as yeast and mammalian cells,is a conventional technique. The vectors are introduced into therecipient cells as pure DNA (transfection) by, for example,precipitation with calcium phosphate (Graham and vander Eb, 1973,Virology 52:466) strontium phosphate (Brash et al., 1987, Mol. Cell.Biol. 7:2013), electroporation (Neumann et al., 1982, EMBO J. 1:841),lipofection (Felgner et al., 1987, Proc. Natl. Acad. Sci USA 84:7413),DEAE dextran (McCuthan et al., 1968, J. Natl. Cancer Inst. 41:351),microinjection (Mueller et al., 1978, Cell 15:579), protoplast fusion(Schafner, 1980, Proc. Natl. Acad. Sci. USA 77:2163-7), or pellet guns(Klein et al., 1987, Nature 327:70). Alternatively, the cDNA can beintroduced by infection with virus vectors, for example retroviruses(Bernstein et al., 1985, Gen. Engrg. 7:235) such as adenoviruses (Ahmadet al., 1986, J. Virol. 57:267) or Herpes (Spaete et al., 1982, Cell30:295).

In view of the many possible embodiments to which the principles of ourdisclosure may be applied, it should be recognized that the illustratedembodiments are only particular examples of the disclosure and shouldnot be taken as a limitation on the scope of the disclosure. Rather, thescope of the disclosure is in accord with the following claims. Wetherefore claim as our invention all that comes within the scope andspirit of these claims.

1. A transformed prokaryotic cell comprising: an exogenous nucleic acid molecule encoding a beta-alanine/pyruvate aminotransferase having at least 95% sequence identity to SEQ ID NO: 20, wherein the beta-alanine/pyruvate aminotransferase is capable of producing malonate semialdehyde and alanine from beta-alanine and pyruvate, and an exogenous nucleic acid molecule encoding an alanine 2,3-aminomutase, wherein the alanine 2,3-aminomutase is capable of producing beta-alanine from alpha-alanine, wherein the prokaryotic cell produces 3-hydroxypropionic acid (3-HP) from beta-alanine.
 2. The transformed cell of claim 1, wherein the exogenous nucleic acid molecule encoding the beta-alanine/pyruvate aminotransferase comprises a sequence having at least 95% sequence identity to SEQ ID NO:
 19. 3. The transformed cell of claim 1, wherein the exogenous nucleic acid molecule encoding the beta-alanine/pyruvate aminotransferase comprises SEQ ID NO:
 19. 4. The transformed cell of claim 1, wherein the cell further comprises dehydrogenase activity capable of converting malonate semialdehyde to 3-HP.
 5. The transformed cell of claim 4, wherein the cell further comprises an exogenous nucleic acid molecule encoding a dehydrogenase capable of converting malonate semialdehyde to 3-HP.
 6. The transformed cell of claim 5, wherein the dehydrogenase is a 3-hydroxypropionate dehydrogenase.
 7. The transformed cell of claim 6, wherein the exogenous nucleic acid molecule encoding the 3-hydroxypropionate dehydrogenase comprises a sequence having at least 95% sequence identity to SEQ ID NO:
 27. 8. The transformed cell of claim 7, wherein the exogenous nucleic acid molecule encoding the 3-hydroxypropionate dehydrogenase comprises SEQ ID NO:
 27. 9. The transformed cell of claim 6, wherein the 3-hydroxypropionate dehydrogenase comprises SEQ ID NO:
 28. 10. The transformed cell of claim 1, wherein the exogenous nucleic acid molecule that encodes an alanine 2,3-aminomutase comprises a sequence having at least 95% sequence identity to SEQ ID NO: 25 and the alanine 2,3-aminomutase is capable of producing beta-alanine from alpha-alanine.
 11. The transformed cell of claim 10, wherein the exogenous nucleic acid molecule that encodes an alanine 2,3-aminomutase comprises SEQ ID NO:
 25. 12. The transformed cell of claim 1, wherein the alanine 2,3-aminomutase comprises SEQ ID NO:
 26. 13. The transformed cell of claim 1, wherein the prokaryotic cell is a Lactobacillus, Lactococcus, Bacillus, or Escherichia cell.
 14. The transformed cell of claim 1, wherein the cell further comprises lipase or esterase activity, or a combination thereof.
 15. The transformed cell of claim 14, wherein the cell further comprises an exogenous nucleic acid molecule encoding a lipase or an esterase.
 16. The transformed cell of claim 1, wherein the cell further comprises: 3-hydroxypropionate dehydrogenase activity and lipase or esterase activity.
 17. The transformed cell of claim 14, wherein the transformed cell produces an ester of 3-HP.
 18. The cell of claim 17, wherein the ester of 3-HP is methyl 3-hydroxypropionate, ethyl 3-hydroxypropionate, propyl 3-hydroxypropionate, butyl 3-hydroxypropionate, or 2-ethylhexyl 3-hydroxypropionate.
 19. The transformed cell of claim 1, wherein the cell further comprises aldehyde dehydrogenase activity and alcohol dehydrogenase activity.
 20. The transformed cell of claim 19 wherein the cell further comprises an exogenous nucleic acid molecule encoding an aldehyde dehydrogenase and an exogenous nucleic acid molecule encoding an alcohol dehydrogenase.
 21. The transformed cell of claim 1, wherein the cell further comprises: 3-hydroxypropionate dehydrogenase activity; aldehyde dehydrogenase activity; and alcohol dehydrogenase activity.
 22. The transformed cell of claim 19, wherein the transformed cell produces 1,3-propanediol.
 23. The transformed cell of claim 1, wherein the cell further comprises esterase activity.
 24. The transformed cell of claim 23, wherein the cell further comprises an exogenous nucleic acid molecule encoding an esterase.
 25. The transformed cell of claim 1, wherein the cell further comprises: 3-hydroxypropionate dehydrogenase activity; and esterase activity.
 26. The transformed cell of claim 23, wherein the transformed cell produces polymerized 3-HP.
 27. A method for making 3-HP from beta-alanine, comprising culturing the transformed cell of claim 1 under conditions that allow the transformed cell to make 3-HP from beta-alanine.
 28. The method of claim 27, wherein the cell is an E. coli cell.
 29. A method of producing an ester of 3-HP, comprising culturing the transformed cell of claim 14 under conditions wherein the transformed cell produces an ester of 3-HP.
 30. The method of claim 29, wherein the ester of 3-HP is methyl 3-hydroxypropionate, ethyl 3-hydroxypropionate, propyl 3-hydroxypropionate, butyl 3-hydroxypropionate, or 2-ethylhexyl 3-hydroxypropionate.
 31. A method of producing 1,3 propanediol, comprising culturing the transformed cell of claim 19 under conditions wherein the transformed cell produces 1,3 propanediol.
 32. A method of producing polymerized 3-HP, comprising culturing the transformed cell of claim 23 under conditions wherein the transformed cell produces polymerized 3-HP.
 33. A method for making 3-HP, comprising: culturing the transformed cell of claim 1 to allow the transformed cell to make 3-HP.
 34. The transformed cell of claim 1, wherein the alanine 2,3-aminomutase comprises at least 95% sequence identity to SEQ ID NO: 26 and is capable of producing beta-alanine from alpha-alanine.
 35. The transformed cell of claim 1, wherein the cell does not express lactate dehydrogenase.
 36. The transformed cell of claim 1, wherein the cell is an E. coli cell.
 37. The transformed cell of claim 1, wherein the exogenous nucleic acid molecule encoding the beta-alanine/pyruvate aminotransferase comprises a sequence that can hybridize under highly stringent hybridization conditions to SEQ ID NO: 19, wherein the highly stringent hybridization conditions comprise incubation at about 42° C. in a hybridization solution containing 25 mM KPO₄ (pH 7.4), 5×SSC, 5×Denhart's solution, 50 μg/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 1-15 ng/mL probe and washes are performed at about 65° C. with a wash solution containing 0.2×SSC and 0.1% SDS.
 38. The transformed cell of claim 1, wherein the exogenous nucleic acid molecule encoding the alanine 2,3-aminomutase comprises a sequence that can hybridize under highly stringent hybridization conditions to SEQ ID NO: 25, wherein the highly stringent hybridization conditions comprise incubation at about 42° C. in a hybridization solution containing 25 mM KPO₄ (pH 7.4), 5×SSC, 5×Denhart's solution, 50 μg/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 1-15 ng/mL probe and washes are performed at about 65° C. with a wash solution containing 0.2×SSC and 0.1% SDS. 